Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy

ABSTRACT

The present disclosure relates in some aspects to methods, cells, and compositions for preparing cells and compositions for genetic engineering and cell therapy. Provided in some embodiments are streamlined cell preparation methods, e.g., for isolation, processing, incubation, and genetic engineering of cells and populations of cells. Also provided are cells and compositions produced by the methods and methods of their use. The cells can include immune cells, such as T cells, and generally include a plurality of isolated T cell populations or types. In some aspects, the methods are capable of preparing of a plurality of different cell populations for adoptive therapy using fewer steps and/or resources and/or reduced handling compared with other methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a US National Stage of International Application No.PCT/US2015/027401 filed 23 Apr. 2015, which claims priority from U.S.provisional application No. 61/983,415, filed Apr. 23, 2014, thecontents of these applications are incorporated by reference in theirentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled735042000200 SeqList.txt, created Oct. 19, 2016, which is 12,952 bytesin size. The information in the electronic format of the SequenceListing is incorporated by reference in its entirety.

FIELD

The present disclosure relates in some aspects to methods, cells, andcompositions for preparing cells, and compositions for geneticengineering and cell therapy. Provided in some embodiments arestreamlined cell preparation methods, e.g., for isolation, processing,incubation, and genetic engineering of cells and populations of cells.Also provided are cells and compositions produced by the methods andmethods of their use. The cells can include immune cells, such as Tcells, and generally include a plurality of isolated T cell populationsor T cell sub-types. In some aspects, the methods are capable ofpreparing of a plurality of different cell populations for adoptivetherapy using fewer steps and/or resources and/or reduced handlingcompared with other methods.

BACKGROUND

Various methods are available for preparing cells for therapeutic use.For example, methods are available for isolating, processing, andengineering cells, including T cells and other immune cells. Methods areavailable to isolate such cells and to express genetically engineeredantigen receptors, such as high affinity T cell receptors (TCRs) andchimeric antigen receptors (CARs). Methods are available to adoptivelytransfer such cells into subjects. Improved methods are needed for thepreparation (e.g., isolation, processing, culturing, and engineering) ofcells for use in cell therapy. In particular, methods are needed for thepreparation and engineering of cells, e.g., a plurality of isolated celltypes or sub-types, with improved efficiency, safety, variability, andconservation of resources. Provided are methods, cells, compositions,kits, and systems that meet such needs.

SUMMARY

Provided are methods for the preparation and engineering of cells andpopulations of cells and cells and compositions produced by the methods.In some embodiments, the cells can be used for immunotherapy, such as inconnection with adoptive immunotherapy methods. In some aspects, theprovided methods include isolation, selection or enrichment of CD4+ andCD8+ cells, or sub-populations thereof, from the same starting sample,such as a single sample, for example a single apheresis sample,leukapheresis sample or a sample containing peripheral blood mononuclearcells (PBMCs). In some embodiments, the methods include selection orenrichment of at least two populations of cells, such as a CD4+ cellpopulation and a CD8+ population, in a single processing stream in whichno negative fraction sample is discarded from a first selection orenrichment of one of the CD4+ or CD8+ in the process, prior toperforming the second selection for the other of the CD4+ or CD8+ cells.In some embodiments, the first and second selection can occursimultaneously or sequentially.

In some aspects, the selection, enrichment and/or isolation of bothpopulations of cells, such as CD4+ and CD8+ cells, is performedsimultaneously, such as in the same vessel or using the same apparatus,or sequentially as part of a system or apparatus in which vessels, e.g.columns, chambers, used in performing the first and second selection,are operably connected. In some embodiments, the simultaneous and/orsequential enrichments or selections can occur as a single processstream without handling of any of the positive or negative fractionsprepared as part of the first and/or second selection or enrichment. Insome aspects, the isolation, culture, and/or engineering of thedifferent populations is carried out from the same starting compositionor material, such as from the same sample.

In some embodiments, the methods include performing a first selection byenriching from a sample containing primary human T cells one of CD4+ orCD8+ cells to generate a first selected population and a non-selectedpopulation, and from the non-selected population performing a secondselection by enriching for the other of CD4+ cell or CD8+ cell, whereinthe method produces a composition of cells containing cells enriched forCD4+ cells and cells enriched for CD8+.

In some embodiments, the second selection is carried out by enrichingfor the other of the T cell subtypes from the non-selected populationgenerated by the first selection. Thus, in some embodiments, theprovided methods differ from other selection methods in that thenegative fraction from a first selection is not discarded but rather isused as the basis for a further selection to enrich for another celltype. In general, where the T cell subset enriched for in the firstselection is a CD4+ subset (or where the first selection enriches forCD4+ cells), it will follow that the first selection is designed suchthat it does not enrich for cells of the other subtype to be enrichedfor in the second selection. For example, in some embodiments, the firstselection enriches for CD4+ cells and does not enrich for CD8+ cells,and the second selection enriches for CD8+ cells from the negativefraction recovered from the first selection. Likewise, in general, wherethe T cell subset enriched for in the first selection is a CD8+ subset(or where the first selection enriches for CD8+ cells), it will followthat the first selection is designed such that it does not enrich forcells of the other subtype to be enriched for in the second selection.For example, in some embodiments, the first selection enriches for CD8+cells and does not enrich for CD4+ cells, and the second selectionenriches for CD4+ cells from the negative fraction recovered from thefirst selection.

In some embodiments, the methods further involve third, fourth, andso-forth further selections, which may enrich for cells from theselected and/or the non-selected populations from any previous selectionstep. For example, in some embodiments, cells from either the selectedpopulation or the non-selected population from a given step (e.g., thesecond selection step, are further enriched. For example, in someembodiments, selected CD8+ cells are further enriched for a subtype ofCD8+ cells, such as resting cells or central memory cells.

In some embodiments, provided is a method for producing geneticallyengineered T cells, comprising (a) providing a culture-initiationcomposition, said composition produced by performing a first selectionin a closed system, said first selection comprising enriching for one ofCD4⁺ cells and CD8+ cells from a sample containing primary human Tcells, thereby generating a first selected population and a non-selectedpopulation, and performing a second selection in the closed system, saidsecond selection comprising enriching for the other of CD4+ cells andCD8+ cells from the non-selected population, thereby generating a secondselected population; (b) incubating a culture-initiating composition,which comprises cells of the first selected population and cells of thesecond selected population, in a culture vessel under stimulatingconditions, thereby generating stimulated cells; and (d) introducing agenetically engineered antigen receptor into stimulated cells generatedin (b), wherein the method thereby generates an output compositioncomprising CD4⁺ T cells and CD8⁺ T cells expressing the geneticallyengineered antigen receptor.

Also provided, in some embodiments, are methods that include asimultaneous enrichment or selection of a first and second population ofcells, such as a CD4+ and CD8+ cell population. In some embodiments, themethod includes contacting cells of a sample containing primary human Tcells with a first immunoaffinity reagent that specifically binds to CD4and a second immunoaffinity reagent that specifically binds to CD8 in anincubation composition, under conditions whereby the immunoaffinityreagents specifically bind to CD4 and CD8 molecules, respectively, onthe surface of cells in the sample, and recovering cells bound to thefirst and/or the second immunoaffinity reagent, thereby generating anenriched composition comprising CD4+ cells and CD8+ cells. In someembodiment, the methods are performed to include in the incubationcomposition a concentration of the first and/or second immunoaffinityreagent that is at a sub-optimal yield concentration so that theenriched composition contains less than 70%, less than 60%, less than50%, less than 40%, less than 30%, less than 20% or less of the totalCD4+ cells in the incubation composition or less than 70% less than 60%,less than 50%, less than 40%, less than 30%, less than 20% or less ofthe CD8+ cells in the incubation composition.

In some embodiments, provided is a method for enriching CD4+ and CD8+ Tcells, comprising providing an enriched composition of CD4+ and CD9+ Tcells, said enriched composition produced by contacting cells of asample containing primary human T cells with a first immunoaffinityreagent that specifically binds to CD4 and a second immunoaffinityreagent that specifically binds to CD8 in an incubation composition,under conditions whereby the immunoaffinity reagents specifically bindto CD4 and CD8 molecules, respectively, on the surface of cells in thesample; and recovering cells bound to the first and/or the secondimmunoaffinity reagent, thereby generating an enriched compositioncomprising CD4+ cells and CD8+ cells at a culture-initiating ratio,wherein: the first and/or second immunoaffinity reagent are present inthe incubation composition at a sub-optimal yield concentration, wherebythe enriched composition contains less than 70% of the total CD4+ cellsin the incubation composition and/or less than 70% of the CD8+ cells inthe incubation composition, thereby producing a composition enriched forCD4+ and CD8+ T cells.

In some embodiments of any of such provided embodiments, the methods areperformed by immunoaffinity-based selection, such as by contacting cellswith an antibody that specifically binds a cell surface marker, such asCD4, CD8 or other cell surface marker, such as expressed on nave,resting or central memory T cells. In some embodiments, the solidsupport is a sphere, such as a bead, such as a microbead or nanobead. Insome embodiments, the bead can be a magnetic bead. In some embodiments,the solid support can be a column or other vessel to effect columnchromatography.

In some embodiments, the antibody contains one or more binding partnerscapable of forming a reversible bond with a binding reagent immobilizedon the solid surface, such as a sphere or chromatography matrix, whereinthe antibody is reversibly mobilized to the solid surface. In someembodiments, cells expressing a cell surface marker bound by theantibody on said solid surface are capable of being recovered from thematrix by disruption of the reversible binding between the bindingreagent and binding partner. In some embodiments, the binding reagent isstreptavidin or is a streptavidin analog or mutant, such as astreptavidin, analog or mutant set forth in any of SEQ ID NOS:11-16 or asequence of amino acids that exhibits at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence setforth in any of SEQ ID NOS: 11-16 and retains binding to a bindingpartner, such as biotin or peptide. In some embodiments, the bindingpartner is biotin, a biotin analog or a peptide capable of binding tothe binding reagent. In some embodiments, the binding partner is orcontains a peptide capable of binding the binding reagent, such as astreptavidin binding peptide, such as a peptide containing the sequenceset forth in any of SEQ ID NOS: 1-10. In some embodiments, the methodsinclude after contacting cells in the sample to the solid supportcontaining the antibody immobilized thereon as part of the first and/orsecond selection, applying a competition reagent to disrupt the bondbetween the binding partner and binding reagent, thereby recovering theselected cells from the solid surface. In some embodiments, thecompetition reagent is biotin or biotin analog.

In some embodiments, the methods generate a selected or enrichedcomposition containing selected or enriched CD4+ cells to CD8+ cells, orsub-populations thereof, present at a culture-initiating ratio. In someembodiments, the culture-initiating ratio of CD4+ to CD8+ cells isbetween at or about 10:1 and at or about 1:10, between at or about 5:1and at or about 1:5 or is between at or about 2:1 and at or about 1:2,such as is at or about 1:1. It is within the level of a skilled artisanto choose or select a sufficient amount or a sufficient relative amountof immunoaffinity-based reagent, such as antibody-coated beads (e.g.magnetic beads) or an affinity chromatography matrix or matrices, toachieve or produce a culture-initiating ratio in the generatedcomposition, such as culture-initiation composition, containing cellsenriched or selected for CD4, CD8 or sub-populations thereof. Exemplaryof such methods are described in subsections below.

In some embodiments, the cells include cells of the immune system, suchas lymphocytes, e.g., T cells (e.g., CD4+ and CD8+ T cells and isolatedsubpopulations thereof), and NK cells. In some embodiments, the cellsare present in combinations of multiple cell populations or cell types,which in some aspects are included in the compositions at particularratios or numbers of the cells or cell types. Also provided are methodsfor optimizing the methods, such as by selecting or determiningappropriate ratios and numbers, such as culture-initiating ratios anddesired output ratios and doses, of the cell types and population, foruse in connection with the methods.

Also provided are cells, cell populations, and compositions thereof, foruse in and produced by the methods. Also provided are systems, devices,apparatuses, reagents, compounds, and kits for carrying out the methods.Also provided are therapeutic methods and uses for cells andcompositions produced by the methods, such as methods for adoptive celltherapy.

In some embodiments, provided are methods for producing cells foradoptive cell therapy, methods for producing cells for geneticengineering, and methods for producing genetically engineered cells. Insome aspects, the cells are T cells, such as CD4⁺ and CD8⁺ T cellsand/or sub-types thereof. In some aspects, the cell therapy is T celltherapy.

In some embodiments, the methods are carried out by (a) isolating cellpopulations from a sample, and (b) incubating a culture-initiatingcomposition in a culture vessel containing cells of the isolatedpopulations. In some aspects, the methods further include geneticallyengineering the incubated cells or cells in the culture vessel, such asby (c) introducing a genetically engineered antigen receptor into cellsin the culture vessel.

In some embodiments, the methods are carried out by (a) incubating aculture-initiating composition in a culture vessel containing aplurality of cell populations at a particular culture-initiation ratioor particular number of cells; and (b) genetically engineering thecells, such as by introducing a genetically engineered antigen receptorinto cells in the culture vessel.

In some embodiments, the genetically engineered antigen receptor isintroduced into cells in the culture vessel, such as to different celltypes or sub-populations within the culture vessel, e.g., to CD4⁺ andCD8⁺ cells in the culture vessel.

In some aspects, the methods produce an output composition for geneticengineering or adoptive cell therapy or comprising cells expressing thegenetically engineered antigen receptor.

In some embodiments, the isolation includes or is carried out byisolating a CD4⁺ primary human T cell population and/or a CD8⁺ primaryhuman T cell population from the sample. In some aspects, it includesdepleting or enriching for a sub-population of CD4⁺ cells, and/ordepleting or enriching for a sub-population of CD8⁺ cells. Thus, in someaspects, the culture-initiating composition includes isolated CD4⁺ andCD8⁺ primary human T cells.

In some aspects, the enriching or depleting is carried out byimmunoaffinity-based selection, such as binding to antibodies or otherbinding molecules recognizing surface markers on the cells. In someaspects, the antibody or other molecule is coupled to a magneticallyresponsive or magnetic particle, e.g., bead. In some aspects, theselection includes positive and/or negative selection steps.

In some embodiments, the isolation of the CD8⁺ primary human T cellpopulation comprises depleting or enriching for a sub-population of CD8⁺cells. In some embodiments, the isolation of the CD4⁺ primary human Tcell population comprises depleting or enriching for a sub-population ofCD4⁺ cells. In some aspects, the isolation of a T cell populationcomprises enriching for T_(CM) cells. In some aspects, the isolation ofthe CD8⁺ and/or the CD4⁺ primary human T cell population comprisesenriching for central memory T (T_(CM)) cells. In some aspects, theenrichment for central memory T (T_(CM)) cells comprises negativeselection for cells expressing a surface marker present on nave T cells,such as CD45RA, or positive selection for cells expressing a surfacemarker present on central memory T cells and not present on nave Tcells, such as CD45RO; and/or positive selection for cells expressingsurface marker present on central memory T (T_(CM)) cells and notpresent on another memory T cell sub-population, such as CD62L, CCR7,CD27, CD127, and/or CD44.

In some embodiments, the isolation includes (i) subjecting the sample topositive selection based on surface expression of CD4, resulting in apositive and first negative fraction, where the positive fraction is theisolated CD4⁺ population; and (ii) subjecting the first negativefraction to negative selection based on surface expression of a non-Tcell marker and a surface marker present on nave T cells, therebygenerating a second negative fraction; and (iii) subjecting the secondnegative fraction to positive selection based on surface expression of amarker present on the surface of central memory T (T_(CM)) cells and notpresent on the surface of another memory T cell sub-population.

In some aspects, the marker present on nave T cells includes CD45RA. Insome aspects, the surface marker present on central memory T (T_(CM))cells and not present on another memory T cell sub-population includesCD62L, CCR7, CD27, CD127, and/or CD44.

In some aspects, the isolation or selections are carried out in the sameseparation vessel. In some aspects, the isolation comprises (i)subjecting the sample to a first selection, thereby generating one ofthe CD4⁺ and CD8⁺ primary human T cell populations and a non-selectedsample; and (ii) subjecting the non-selected sample to a secondselection, thereby generating the other of the CD4⁺ and CD8⁺ primaryhuman T cell populations. In some aspects, the CD4⁺ primary human T cellpopulation is generated in the first selection and the CD8⁺ primaryhuman T cell population is generated in the second selection. In someaspects, the first and/or second selection comprises a plurality ofpositive or negative selection steps.

In some aspects, the isolation of one or more of the populations, suchas the primary human T cell population, the primary human CD4+ T cellpopulation, or the primary human CD8+ T cell population, includespositive selection based on surface expression CD62L, CCR7, CD44, orCD27. In some aspects, the isolation of one or more of the populations,such as the primary human T cell population, the primary human CD4⁺ Tcell population, or the primary human CD8⁺ T cell population, includesnegative selection based on surface expression of CD45RA or positiveselection based on surface expression of CD45RO.

In some embodiments, the various isolations, such as isolation of theplurality of cell populations, e.g., the CD4⁺ and the CD8⁺ populations,are carried out in the same separation vessel. In some aspects, theseparation vessel is or includes a tube, tubing set, chamber, unit,well, culture vessel, bag, and/or column. In some aspects, theseparation vessel maintains the cells in a contained or sterileenvironment during the separation.

In some embodiments, the incubating is carried out under stimulatingconditions. In some aspects, the culture-initiating compositioncomprises the CD4⁺ and CD8⁺ primary human T cell populations at aculture-initiating ratio. In some aspects, the culture-initiating ratiois designed to yield a desired output ratio of CD4⁺ to CD8⁺ cells (ordesired total number of T cells or number(s) of the sub-population(s))following said incubation, or at some later time, such as followingengineering, cryopreservation, or at the time just prior toadministration, such as at thaw at bedside.

In some embodiments, the desired output ratio is between at or about 5:1and at or about 1:5 (or greater than about 1:5 and less than about 5:1),such as between at or about 1:3 and at or about 3:1 (or greater thanabout 1:3 and less than about 3:1), such as between at or about 2:1 andat or about 1:5 (or greater than about 1:5 and less than about 2:1), oris the range of between at or about 2:1 and at or about 1:5. In someaspects, the desired output ratio is at or about 3:1, 2.9:1, 2.8:1,2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2:1, 1.9:1, 1.8:1,1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2,1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5,1:4, 1:4.5, or 1:5.

In some aspects, the method results in a ratio of two different celltypes or populations, such as a ratio of CD4⁺ to CD8⁺ cells, in theoutput composition that is between at or about 5:1 and at or about 1:5(or greater than about 1:5 and less than about 5:1), such as between ator about 1:3 and at or about 3:1 (or greater than about 1:3 and lessthan about 3:1), such as between at or about 2:1 and at or about 1:5 (orgreater than about 1:5 and less than about 2:1), or that is at or about3:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2:1,1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1,1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2,1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.

In some aspects, the desired output ratio is 1:1 or is about 1:1.

In some aspects, the methods result in the desired output ratio or cellnumber(s) in the output composition, results in a ratio or number in theoutput composition that is within a certain tolerated difference orrange of error of such a desired output ratio or number, and/or resultsin such a ratio or number a certain percentage of the time that themethod is performed, such as at least at or about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more than 95% of the time.

In some aspects, the tolerated difference is within about 1%, about 2%,about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50% of the desiredratio. In some aspects, the output ratio is within 20% of the desiredratio and/or is within that ratio at least 80% of the time the methodsare performed.

In some embodiments, the tolerated difference and/or the desired outputratio or number(s) is or has been determined by administering differentcell types, such as administering CD4⁺ and CD8⁺ cells, to one or moresubjects at a plurality of test ratios or numbers, and assessing one ormore parameters. In some aspects, the determination of the desiredoutput ratio or number or tolerated difference includes assessing one ormore outcomes following administration to the subject. In some aspects,the outcomes include those selected from among amelioration of a diseasesymptom and outcomes indicating safety and/or low or absence oftoxicity.

In some embodiments, the culture-initiation ratio is selected based on aproliferation rate or survival capacity of various cell types isolated,such as the CD4⁺ and/or CD8⁺ primary human T cell population. In someembodiments, the culture-initiating ratio is selected based on a sourceof the sample, such as the subject from which the sample has beenderived. For example, in some aspects, the sample is derived from asubject and the culture-initiating ratio is selected based on a diseaseor condition affecting said subject and/or a treatment the subject isreceiving, has received, or will receive, such as a co-treatment foradministration with the adoptive cell therapy. In some aspects, theculture-initiating ratio is selected based on a phenotype of one or moreof the cells or sub-types of cells being isolated or cultured, such as aphenotype of the CD4+ and/or CD8+ cell populations, such as expressionof a cell surface marker or synthesis or secretion of one or morefactors, such as cytokines or chemokines.

In some embodiments, the methods comprise selecting theculture-initiation ratio prior to the incubation step. In some aspects,the selection is carried out by measuring a proliferation rate orsurvival capacity of one or more of the isolated cell populations orpopulations of cells being incubated, such as the CD4⁺ primary human Tcells and/or the isolated CD8⁺ primary human T cells. In some aspects,the selection is carried out by assessing a phenotype of the isolatedCD4⁺ primary human T cells and/or the isolated CD8⁺ primary human Tcells. In some aspects, the phenotype selected from among expression ofa surface marker and secretion of a cytokine or other factor. In someaspects, the selecting is carried out by assessing the source of thesample, such as where the culture-initiation ratio is selected based ona disease or condition affecting a subject from which the sample isderived.

In some embodiments, the methods further include determining anintermediate ratio or number of cells, such as an intermediate ratio ofCD4⁺ to CD8⁺ cells, present in the culture vessel at a time pointsubsequent to the initiation of said incubation. In some aspects, themethods further include adjusting one or more parameters and/or orincreasing or decreasing the time for carrying out the incubation and/orengineering steps, based on said intermediate ratio. In one aspect, theadjusting comprises increasing or decreasing the number of or enrichingfor one or more cell populations in the culture vessel, such asincreasing or enriching for CD4⁺ or CD8⁺ cells in the culture vessel,adjusting temperature, adding a stimulant to the culture vessel,adjusting the concentration of one or more stimulants in the culturevessel, and/or adding and/or removing a sub-population of cells to orfrom the culture vessel. In some aspects, the determining and/oradjusting are carried out while maintaining the composition beingincubated within a sterile or contained environment. In some aspects,the determination and/or adjusting are carried out in an automatedfashion, such as controlled by a computer attached to a device in whichthe steps are performed.

In some aspects, the isolating, incubating, and/or engineering steps arecarried out in a sterile or contained environment and/or in an automatedfashion, such as controlled by a computer attached to a device in whichthe steps are performed.

In some aspects, the CD8⁺ population in the culture-initiatingcomposition comprises at least 50% central memory T (T_(CM)) cells orcomprises less than 20% nave T (T_(N)) cells.

In some embodiments, the sample is obtained from a subject. In someaspects, the subject is a subject to whom said genetically engineeredcells, e.g., T cells, or cells for adoptive cell therapy will beadministered or subject in need of such administration. In otheraspects, the subject is a subject other than a subject to whom saidgenetically engineered cells, e.g., T cells, or cells for adoptivetherapy will be administered or is a subject not in need of suchtherapy. Among the samples are blood and blood-derived samples, such aswhite blood cell samples, apheresis samples, leukapheresis samples,peripheral blood mononuclear cell (PBMC) samples, and whole blood.

In some aspects, the stimulating conditions for the incubation orengineering include conditions whereby T cells of the culture-initiatingcomposition proliferate or expand. For example, in some aspects, theincubation is carried out in the presence of an agent capable ofactivating one or more intracellular signaling domains of one or morecomponents of a TCR complex, such as a CD3 zeta chain, or capable ofactivating signaling through such a complex or component. In someaspects, the incubation is carried out in the presence of an anti-CD3antibody, and anti-CD28 antibody, anti-4-1BB antibody, for example, suchantibodies coupled to or present on the surface of a solid support, suchas a bead, and/or a cytokine, such as IL-2, IL-15, IL-7, and/or IL-21.

In some embodiments, the genetically engineered antigen receptor is orincludes a T cell receptor (TCR), such as a high-affinity TCR, orfunctional non-TCR antigen receptor, such as a chimeric antigen receptor(CAR). In some aspects, the receptor specifically binds to an antigenexpressed by cells of a disease or condition to be treated. In someaspects, the CAR contains an extracellular antigen-recognition domain.In some aspects, it further contains an intracellular signaling domaincomprising an ITAM-containing sequence and an intracellular signalingdomain of a T cell costimulatory molecule.

Also provided are cells and compositions, including pharmaceuticalcompositions, produced by any of the methods or embodiments, includinggenetically engineered cells and cells for adoptive cell therapy. Alsoprovided are methods for administering such cells and compositions tosubjects and uses of the cells and compositions in such methods. Forexample, provided are methods of treatment carried out by producingcells according to the cell production methods and administering cellsof the output composition or a composition derived therefrom to asubject. Provided are methods of treatment including administering theprovided cells or compositions to a subject. In some aspects, the samplefrom which the cells are isolated is derived from the subject to whichthe cells are administered. In some aspects, the sample is from adifferent subject. Thus, the methods include autologous and allogeneicmethods. In some embodiments, the methods ameliorate, treat, or preventone or more symptoms of a disease or condition in the subject. In someaspects, the disease or condition is a cancer or associated symptom. Insome embodiments, the cancers include leukemia, lymphoma, e.g., chroniclymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acute myeloidleukemia, multiple myeloma, refractory follicular lymphoma, mantle celllymphoma, indolent B cell lymphoma, B cell malignancies, cancers of thecolon, lung, liver, breast, prostate, ovarian, skin (includingmelanoma), bone, and brain cancer, ovarian cancer, epithelial cancers,renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma,cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma,Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/ormesothelioma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: provides a schematic representation of an embodiment of aclosed system for use in embodiments of the provided methods. Thedepicted exemplary system includes a cell sample 5, a washing bufferreservoir 6, an elution buffer reservoir 7, a first Fab reservoir 18, asecond Fab reservoir 19, and a pump 8, connected through a series oftubing and valves 13, to a first chromatography column 1 containing afirst matrix 3 operably linked via a series of tubing lines to a secondchromatography column 2 containing a second matrix 4. The secondchromatography column 2 is operably linked to a removal chamber 9. Theremoval chamber 9 is operably linked to a valve 13, which directs cellsand fluids to a waste container 10 or a culture vessel 12 via a seriesof tubing lines. The system is enclosed in an enclosure 14.

FIG. 1B: provides a schematic representation of an embodiment of aclosed system for use in embodiments of the provided methods. Thedepicted exemplary system includes a cell sample 5, a washing bufferreservoir 6, an elution buffer reservoir 7, a first Fab reservoir 18, asecond Fab reservoir 19, a third Fab reservoir 20, and a pump 8,connected through a series of tubing to a first chromatography column 1containing a first matrix 3. Valves 13 operably linked to the series oftubing direct fluids through the series of tubing. A valve 13 operablylinked to the first chromatography column 1 directs cells and fluids toa second chromatography column 2 containing a second matrix 4, which isoperably linked to a removal chamber 9. The valve 13 operably linked tothe first chromatography column 1 also directs cells and fluids to aremoval chamber 9, which is operably linked to a third chromatographycolumn 15 containing a third matrix 16. The third chromatography column15 is operably linked to a removal chamber 9. The removal chambers 9 areoperably linked to a first waste container 10 or a culture vessel 12 viathe series of tubing lines and valves 13. The system is enclosed in anenclosure 14.

DETAILED DESCRIPTION

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. METHODS AND SYSTEMS FOR ISOLATING, CULTURING, AND ENGINEERING CELLSFOR ADOPTIVE THERAPY

Provided are methods of preparing cells, e.g., T cells, for use ingenetic engineering and therapeutic methods such as adoptive celltherapy. Specifically, in some embodiments, the methods use or generatecompositions that contain a plurality of different cell populations ortypes of cells, such as isolated CD4⁺ and CD8⁺ T cell populations andsub-populations. In some embodiments, the methods include steps forisolating one or more cell populations, generally a plurality of cellpopulations. The cells generally are isolated from a sample derived froma subject.

In some aspects, the methods are performed by employing simultaneous orsequential selections or enrichments in which a plurality of differentcell populations, such as CD4+ or CD8+ cells, from a sample, such as asample containing primary human T cells, are selected, enriched and/orisolated. In some embodiments, such methods are performed using a singleprocessing stream in which a first population of cells, such as CD4+ orCD8+ cells, and a second population of cells, such as the other of theCD4+ or CD8+ cells, are selected, enriched and/or isolated withoutdiscarded any cells from the first population prior to performing theselection of the second population of cells. In some aspects, the firstand second selections can be performed simultaneously or sequentially.In some embodiments, the methods of selection are performed as a singleprocess stream by performing a first selection to enrich for a firstpopulation of cells, such as one of CD4+ and CD8+ cells from a sample,such as a sample containing primary human T cells, and using thenon-selected cells from the first selection as the source of cells for asecond selection, such as for the other of the CD4+ or CD8+ cells fromthe sample.

In some embodiments, a further selection or selections can be performedof the first or second selected cells. In some embodiments, the furtherselection enriches for a sub-population of CD4+ or CD8+ cells expressinga marker on central memory T (T_(CM)) cells and/or enriches for asub-population of cells expressing CD62L, CD45RA, CD45RO, CCR7, CD27,CD127, or CD44.

In some embodiments, the methods of selection are performed in a closedsystem or apparatus. In some embodiments, within the closed system orapparatus, a composition, such as a culture-initiation composition, isgenerated containing the enriched or selected population of cells, suchas both the enriched or selected CD4+ and CD8+ populations, in the samecomposition.

For example, in some aspects, the one or more steps are carried out withthe plurality of cell populations combined in the same composition, orpresent in the same vessel, e.g., in the same closed system orapparatus, or in the same vessel, unit, or chamber, such as the samecolumn, e.g., magnetic separation column, tube, tubing set, culture orcultivation chamber, culture vessel, processing unit, cell separationvessel, centrifugation chamber, or using the same separation matrix,media, and/or reagents, such as the same magnetic or magneticallyresponsive matrix, particle, or bead, the same solid support, e.g.,affinity-labeled solid support, and/or the same antibodies and/or otherbinding partners, such as fluorescently-labeled antibodies and bindingpartners, for the plurality of cell populations. In some embodiments,the one or more vessels, units or chambers, such as columns, areoperably connected in the same closed system or apparatus, so that themethods of isolation, selection and/or enriching occurs in a singleprocess stream in which a non-selected cell population from a firstselection can be used as a source of cells for a second selection.

In some embodiments, the isolation of or enrichment for one or morespecific populations or sub-populations of cells, e.g., CD4+ and CD8+ Tcells to be cultured or engineered for adoptive therapy, provides one ormore advantages. For example, engineering cells enriched for a pluralityof different cell populations or types of cells, such as isolated CD4⁺and CD8⁺ T cell populations and sub-populations, can improve efficacy ofor reduce or avoid unwanted effects. In some aspects, the isolation orenrichment increases the ability of cells ultimately administered to asubject to persist, expand, become activated, and/or engraft in vivo orupon administration to a subject. In some aspects, it improves orincreases one or more effector function or activation phenotype. Forexample, in some aspects, enriching a T cell population, such as a CD8+T cell population, for central memory (T_(CM)) cells can provide suchadvantages. In some aspects, one or more of such advantages are providedby combining two or more isolated populations or sub-types, such asisolated populations or sub-populations of CD8+ and CD4+ T cells, suchas a T_(CM)-enriched CD8⁺ population and a CD4⁺ population. For example,such advantages can be achieved in some aspects by administering a CD4+and a CD8+ population in comparison with a CD8+ population alone.

The methods in some embodiments include processing of a generatedcomposition containing a plurality of the isolated or selected cellpopulations, such as a population of selected or enriched CD4+ cells andCD8+ cells. In one embodiment, processing of the generated compositionincludes incubating the cells under stimulating conditions, for examplein some aspects, to activate the cells for engineering or transductionor for cell expansion. The methods, in some embodiments, include stepsfor engineering a plurality of cell types, such as CD4+ cells and CD8+cells, such as those isolated and present in the incubated composition,such as the culture-initiation composition. In some aspects, theengineering is carried out to introduce a genetically engineered antigenreceptor into the cells, such as a TCR, e.g., a high-affinity TCR, or afunctional non-TCR antigen receptor, such as a chimeric antigen receptor(CAR). In some aspects, the methods include further processing, such asfurther incubation, for example at or about 37° C.±2° C., and/orformulating of cells and compositions containing the same. In someembodiments, the processing produces a resulting output compositioncontaining genetically engineered cells, such as genetically engineeredCD4+ cells and CD8+ cells. In some embodiments, the resulting processedoutput composition can be used in methods for administering cells andcompositions prepared by the methods to a patient, for example, inconnection with adoptive cell therapy.

In certain embodiments, the isolation or separation is carried out usinga system, device, or apparatus that carries out one or more of theisolation, cell preparation, separation, processing, incubation,culture, and/or formulation steps of the methods. In some aspects, thesystem is used to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inInternational Patent Application, Publication Number WO2009/072003, orUS 20110003380 A1. In some embodiments, the system is a closed apparatusor system, such as is depicted in FIG. 1A or FIG. 1B. In someembodiments, the system or apparatus is automated and/or carries out theselection steps to enrich cells according to the methods in an automatedfashion.

In some embodiments, the system or apparatus carries out the isolation,such as selection or enrichments steps. In some embodiments, a furthersystem or apparatus, such as a closed system or apparatus, can be usedto carry out one or more of the other steps such as cells preparation,processing, incubation, culture and/or formulation steps of the method.In some embodiments, the system or apparatus carries out one or more,e.g., all, of the isolation, processing, engineering, and formulationsteps in an integrated or self-contained system, and/or in an automatedor programmable fashion. In some aspects, the system or apparatusincludes a computer and/or computer program in communication with thesystem or apparatus, which allows a user to program, control, assess theoutcome of, and/or adjust various aspects of the processing, isolation,engineering, and formulation steps.

In some embodiments, because the enriched composition, such as cultureinitiation composition, is generated by the provided methods to containdifferent cell populations, such as CD4+ and CD8+, the generatedcomposition can be processed together and simultaneously under the sameconditions, and in some aspects, in a closed system. Thus, in someaspects, the methods provide a process to produce and process apopulation of selected, enriched or isolated cells containing differentpopulations of cells, such as CD4+ and CD8+ cells, in a single processstream. In some aspects, this differs from prior art methods, includingprior art methods that process cells for engineering for adoptive celltherapy, which typically include a separate process stream for eachdifferent cell population, such as at least two process streams. Forexample, in some aspects of existing methods, CD4+ T cells areseparately isolated, enriched and/or selected and processed understimulating conditions for genetic engineering, CD8+ T cells areseparately isolated, enriched and/or selected and processed understimulating conditions for genetic engineering, and the separateprocessed and engineered CD4+ and CD8+ T cells are re-combined prior toadministration to a subject.

In some embodiments, the methods provide one or more advantages comparedwith other preparation, isolation, incubation, and engineering methods,such as cost, time, and/or resource savings. Such advantages can includethe ability to isolate, process, e.g., incubate, and/or engineer theplurality of cell populations, present at or near a desired ratio, withincreased efficiency and/or reduced complexity, time, cost, and/or useof resources, compared with other methods.

In some embodiments, such advantages are achieved by streamlining one ormore of the method steps. For example, in some aspects, isolation,culture, and/or engineering of the different populations is carried outusing the same apparatus or equipment and/or simultaneously. In someaspects, the isolation, culture, and/or engineering of the differentpopulations is carried out based on the same starting composition. Insome aspects, such features of the methods reduce the amount of time,method steps, cost, complexity, and/or number of resources, compared toa method in which the cell populations are isolated, incubated, and/orengineered separately, in separate vessels, using separate equipment, atseparate times, and/or beginning with different starting compositions.

In some aspects, the methods of isolating, incubating and engineeringcells results in an output composition in which the different cellpopulations or cell types, such as CD4+ cells and CD8+ cells, arepresent at a desired ratio, or within a certain degree of toleratederror of the desired ratio. Such ratios include those deemed optimal forthe therapeutic use, e.g., output ratios deemed appropriate or optimalfor administration to a patient in connection with adoptive celltherapy. Also provided are methods for administering cells andcompositions prepared by the methods to a patient, for example, inconnection with adoptive cell therapy.

In some embodiments, the methods of isolation or selection are performedto achieve selection of cells at a chosen culture-initiation ratio ofCD4+ cells to CD8+ cells or a sub-population thereof. In some aspects,such ratios include ratios deemed optimal starting points for achievingan optimal ratio, such as a desired ratio in the output composition, atthe completion of the method or one or more steps, such as following aculture, incubation, and/or engineering step. In some embodiments,providing the cells at a culture-initiation ratio, such as a ratio ofCD4+ cells to CD8+ cells, accounts for differences in expansion of CD4+and CD8+ T cells that can occur upon stimulation or activation withdifferent reagents, in order to achieve a desired ratio in the outputcomposition.

In some embodiments, the methods generate engineered cells or cells forengineering at or within a certain percentage of a desired output ratio,or do so at least a certain percentage of the time. The desired outputratio typically is a ratio that has been determined to be optimal foradministration to a patient via adoptive transfer. In some embodiments,the populations or sub-types of cells, such as CD8⁺ and CD4⁺ T cells areadministered at or within a tolerated difference of a desired dose oftotal cells, such as a desired dose of T cells. In some aspects, thedesired dose is a desired number of cells or a desired number of cellsper unit of body weight of the subject to whom the cells areadministered, e.g., cells/kg. In some aspects, the desired dose is at orabove a minimum number of cells or minimum number of cells per unit ofbody weight. In some aspects, among the total cells, administered at thedesired dose, the individual populations or sub-types are present at ornear a desired output ratio (such as CD4⁺ to CD8⁺ ratio), e.g., within acertain tolerated difference or error of such a ratio.

In some embodiments, the cells are administered at or within a tolerateddifference of a desired dose of one or more of the individualpopulations or sub-types of cells, such as a desired dose of CD4+ cellsand/or a desired dose of CD8+ cells. In some aspects, the desired doseis a desired number of cells of the sub-type or population, or a desirednumber of such cells per unit of body weight of the subject to whom thecells are administered, e.g., cells/kg. In some aspects, the desireddose is at or above a minimum number of cells of the population orsub-type, or minimum number of cells of the population or sub-type perunit of body weight.

Thus, in some embodiments, the dosage is based on a desired fixed doseof total cells and a desired ratio, and/or based on a desired fixed doseof one or more, e.g., each, of the individual sub-types orsub-populations. Thus, in some embodiments, the dosage is based on adesired fixed or minimum dose of T cells and a desired ratio of CD4⁺ toCD8⁺ cells, and/or is based on a desired fixed or minimum dose of CD4⁺and/or CD8⁺ cells.

In some embodiments, provided are methods for determining such optimaloutput ratios and/or desired doses, and/or levels of variance from thedesired output ratio or desired dose that would be tolerated, e.g., atolerated difference. In some embodiments, in order to achieve thedesired output ratio or dose(s) (or to achieve such a ratio a certainpercentage of the time or within a certain tolerated difference), thecell populations are combined or incubated at ratios (e.g.,culture-initiation ratios) designed to achieve the desired output ratiofor adoptive transfer.

Also provided are methods for determining a culture-initiation ratiodesigned to achieve the desired output ratio or dose to do so within acertain tolerated difference and/or a certain percentage of the time.Also provided are methods for assessing interim ratios or numbers of thecell populations, such as at one or more periods of times over thecourse of the various method steps, e.g., during incubation. Alsoprovided are methods for adjusting various conditions, such as cultureconditions, based on such assessments. In some aspects, the adjustmentsare carried out to ensure that a particular output ratio or dose isachieved or achieved within a tolerated difference.

In particular embodiments, provided are streamlined methods forpreparing a composition having at or near a desired output ratio of aCD4⁺ T cell population and a CD8⁺ T cell population (e.g., a CD8⁺population enriched for a sub-type of T cells such as central memory Tcells), and/or a desired dose (e.g., number or number per unit of bodyweight) of T cells and/or of CD4⁺ and CD8⁺ T cells, for introduction ofa genetically engineered antigen receptor for use in adoptive celltherapy, where the cell populations are isolated, incubated, and/orengineered in combination and the method is associated with increasedefficiency and/or reduced complexity, time, cost, and/or use ofresources compared to a method in which the populations are isolated,incubated, and/or engineered separately.

Also provided are cells and compositions prepared by the methods,including pharmaceutical compositions and formulations, and kits,systems, and devices for carrying out the methods. Also provided aremethods for use of the cells and compositions prepared by the methods,including therapeutic methods, such as methods for adoptive celltherapy, and pharmaceutical compositions for administration to subjects.

A. Isolation, Isolated Cells, and Other Processing Steps

Among the provided embodiments are methods for isolating a plurality ofcells and populations of cells from a sample, as well as isolated, suchas enriched, cells produced by such methods. The isolation can includeone or more of various cell preparation and separation steps, includingseparation based on one or more properties, such as size, density,sensitivity or resistance to particular reagents, and/or affinity, e.g.,immunoaffinity, to antibodies or other binding partners. In someaspects, the isolation is carried out using the same apparatus orequipment sequentially in a single process stream and/or simultaneously.In some aspects, the isolation, culture, and/or engineering of thedifferent populations is carried out from the same starting compositionor material, such as from the same sample.

In some aspects, the plurality of cell populations are isolated in thesame closed system or apparatus, and/or in the same vessel or set ofvessels, e.g., same (or same set of) unit, chamber, column, e.g.,magnetic separation column, tube, tubing set, culture or cultivationchamber, culture vessel, processing unit, cell separation vessel,centrifugation chamber. For example, in some cases, the isolation of aplurality of cell populations is carried out a system or apparatusemploying a single or the same isolation or separation vessel or set ofvessels, such as a single column or set of columns, and/or same tube, ortubing set, for example, without requirements to transfer the cellpopulation, composition, or suspension from one vessel, e.g., tubingset, to another.

In some aspects, such methods are achieved by a single process stream,such as in a closed system, by employing simultaneous or sequentialselections in which a plurality of different cell populations, such asCD4+ or CD8+ cells, from a sample, such as a sample containing primaryhuman T cells, are selected, enriched and/or isolated. In oneembodiment, a sample containing cells is subjected to a selection bysimultaneous enrichment of both the CD4+ and CD8+ populations. In someaspects, carrying out the separation or isolation in the same vessel orset of vessels, e.g., tubing set, is achieved by carrying out sequentialpositive and negative selection steps, the subsequent step subjectingthe negative and/or positive fraction from the previous step to furtherselection, where the entire process is carried out in the same tube ortubing set. In one embodiment, a sample containing cells to be selectedis subjected to a sequential selection in which a first selection iseffected to enrich for one of the CD4+ or CD8+ populations, and thenon-selected cells from the first selection is used as the source ofcells for a second selection to enrich for the other of the CD4+ or CD8+populations. In some embodiments, a further selection or selections canbe effected to enrich for sub-populations of one or both of the CD4+ orCD8+ population, for example, central memory T (T_(CM)) cells.

In a particular aspect, a first selection step is carried out usingbeads labeled with CD4-binding molecules, such as antibodies (orsecondary reagents that recognize such molecules), and the positive andnegative fractions from the first selection step are retained, followedby further positive or negative selection of the negative fraction toenrich for CD8+ cells, such as by using beads labeled with CD8-bindingmolecules, and optionally selection of a sub-population of cells fromthe CD8+ fraction, such as central memory CD8+ T cells and/or cellsexpressing one or more markers CD62L, CD45RA, CD45RO, CCR7, CD27, CD127,or CD44. In some embodiments, the order of the selections can bereversed. In some embodiments, a CD4 selection is always performed firstand, from the negative fraction (CD4−), the CD8+ fraction is enriched ina second selection, e.g., by negative selection for one or more ofCD45RA⁺ and CD14, and/or positive selection for one or more of CD62L,CCR7, and/or other markers expressed on central memory cells.

In some embodiments, a plurality of cell populations, e.g., a CD4⁺ Tcell population and a CD8⁺ T cell population, is isolated, for example,to produce a culture-initiating composition containing the plurality ofcell populations. The culture-initiating composition typically containsthe cells at a culture-initiating ratio, which is designed to yield aparticular desired output ratio of two or more cell types, such as aparticular CD4⁺ to CD8⁺ ratio, following one or more incubation,culture, cultivation, and/or engineering steps. The desired output ratioin some embodiments is a ratio designed to be optimal for administrationof the cells to a patient, e.g., in adoptive cell therapy.

In some aspects, isolating the plurality of populations in a single orin the same isolation or separation vessel or set of vessels, such as asingle column or set of columns, and/or same tube, or tubing set orusing the same separation matrix or media or reagents, such as the samemagnetic matrix, affinity-labeled solid support, or antibodies or otherbinding partners, include features that streamline the isolation, forexample, resulting in reduced cost, time, complexity, need for handlingof samples, use of resources, reagents, or equipment. In some aspects,such features are advantageous in that they minimize cost, efficiency,time, and/or complexity associated with the methods, and/or avoidpotential harm to the cell product, such as harm caused by infection,contamination, and/or changes in temperature.

In some embodiments the isolated cell populations obtained for use inthe methods herein are sterile. Microbial contamination of cellseparation products can in some cases lead to the infection of therecipient subject, such as an immunocompromised recipient patient unableto fight the infection. In some embodiments, the cells, cellpopulations, and compositions are produced under GMP (good manufacturingpractice) conditions. In some embodiments, GMP conditions comprisestringent batch testing. In certain embodiments, tissue typing isperformed prior to transplantation, e.g., to avoid human leukocyteantigen (HLA) mismatch and prevent problems such as graft-versus-hostdisease. In some embodiments, the provided methods reduce handling byindividual users and automate various steps, which in some aspects canincrease consistency of the isolated cell populations and compositionsand reduces error, thereby promoting consistency of the therapy andsafety.

1. Cells and Populations of Cells

In some embodiments, the methods include performing a selection,isolation and/or enrichment of a cell sample, such as a primary humancell sample. The isolated cell populations typically include a pluralityof cell populations, generally populations of blood or blood-derivedcells, such as hematopoietic cells, leukocytes (white blood cells),peripheral blood mononuclear cells (PBMCs), and/or cells of the immunesystem, e.g., cells of the innate or adaptive immunity, such as myeloidor lymphoid cells, e.g., lymphocytes, typically T cells and/or NK cells.In some embodiments, the sample is an apheresis or leukapheresis sample.In some embodiments, the selection, isolation and/or enrichment caninclude positive or negative selection of cells from the sample.

In some embodiments, the sample is a sample containing primary human Tcells, such as CD4+ and CD8+ T cells. In particular embodiments, thesample is one in which a plurality of T cell populations is isolated,such as a population of CD4⁺ cells and a population of CD8⁺ T cells.Thus, in some embodiments, the isolation includes positive selection forcells expressing CD4 or cells expressing CD8 and/or negative selectionfor cells expressing non-T cell markers, such as myeloid or B cellmarkers, for example, negative selection for cells expressing CD14,CD19, CD56, CD20, CD11b, and/or CD16.

In some embodiments, the sample is one containing a plurality ofpopulations that includes a T cell population, such as a whole T cell orCD4+ population, and an NK cell population. Among the T cell populationsthat can be enriched, isolated and/or selected are populations ofunfractionated T cells, unfractionated CD4+ cells, unfractionated CD8+cells, and sub-populations of CD4+ and/or CD8+ T cells, includingsubpopulations of T cells generated by enrichment for or depletion ofcells of a particular sub-type or based on a particular surface markerexpression profile.

For example, among the sub-types of T cells (e.g., CD4⁺ or CD8⁺ T cells)that can be enriched, isolated and/or selected are those defined byfunction, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation.

Among the sub-types and subpopulations of T cells and/or of CD4+ and/orof CD8+ T cells that can be enriched, isolated and/or selected are naveT (TN) cells, effector T cells (TEFF), memory T cells and sub-typesthereof, such as stem cell memory T (TSCM), central memory T (TCM),effector memory T (TEM), or terminally differentiated effector memory Tcells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature Tcells, helper T cells, cytotoxic T cells, mucosa-associated invariant T(MAIT) cells, naturally occurring and adaptive regulatory T (Treg)cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta Tcells, and delta/gamma T cells.

In some embodiments, one or more of the T cell populations enriched,isolated and/or selected from a sample by the provided methods are cellsthat are positive for (marker+) or express high levels (markerhigh) ofone or more particular markers, such as surface markers, or that arenegative for (marker−) or express relatively low levels (markerlow) ofone or more markers. In some cases, such markers are those that areabsent or expressed at relatively low levels on certain populations of Tcells (such as non-memory cells) but are present or expressed atrelatively higher levels on certain other populations of T cells (suchas memory cells). In one embodiment, the cells (such as the CD8+ cellsor the T cells, e.g., CD3+ cells) are enriched for (i.e., positivelyselected for) cells that are positive or expressing high surface levelsof CD45RO, CCR7, CD28, CD27, CD44, CD127, and/or CD62L and/or depletedof (e.g., negatively selected for) cells that are positive for orexpress high surface levels of CD45RA. In some embodiments, cells areenriched for or depleted of cells positive or expressing high surfacelevels of CD122, CD95, CD25, CD27, and/or IL7-Rα (CD127). In someexamples, CD8+ T cells are enriched for cells positive for CD45RO (ornegative for CD45RA) and for CD62L.

In some embodiments, a CD4+ T cell population and a CD8+ T cellsub-population, e.g., a sub-population enriched for central memory (TCM)cells.

In some embodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

2. Samples

The cells and cell populations typically are isolated from a sample,such as a biological sample, e.g., one obtained from or derived from asubject, such as one having a particular disease or condition or in needof a cell therapy or to which cell therapy will be administered. In someaspects, the subject is a human, such as a subject who is a patient inneed of a particular therapeutic intervention, such as the adoptive celltherapy for which cells are being isolated, processed, and/orengineered. Accordingly, the cells in some embodiments are primarycells, e.g., primary human cells. The samples include tissue, fluid, andother samples taken directly from the subject, as well as samplesresulting from one or more processing steps, such as separation,centrifugation, genetic engineering (e.g. transduction with viralvector), washing, and/or incubation. The biological sample can be asample obtained directly from a biological source or a sample that isprocessed. Biological samples include, but are not limited to, bodyfluids, such as blood, plasma, serum, cerebrospinal fluid, synovialfluid, urine and sweat, tissue and organ samples, including processedsamples derived therefrom.

In some aspects, the sample is blood or a blood-derived sample, or is oris derived from an apheresis or leukapheresis product. Exemplary samplesinclude whole blood, peripheral blood mononuclear cells (PBMCs),leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia,lymphoma, lymph node, gut associated lymphoid tissue, mucosa associatedlymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach,intestine, colon, kidney, pancreas, breast, bone, prostate, cervix,testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.Samples include, in the context of cell therapy, e.g., adoptive celltherapy, samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig.

3. Cell Processing, Preparation and Non-Affinity-Based Separation

In some embodiments, isolation of the cells or populations includes oneor more preparation and/or non-affinity based cell separation steps. Insome examples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contains cells other thanred blood cells and platelets.

In some embodiments, the blood cells collected from the subject arewashed, e.g., to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In someembodiments, the cells are washed with phosphate buffered saline (PBS).In some embodiments, the wash solution lacks calcium and/or magnesiumand/or many or all divalent cations. In some aspects, a washing step isaccomplished a semi-automated “flow-through” centrifuge (for example,the Cobe 2991 cell processor, Baxter) according to the manufacturer'sinstructions. In some aspects, a washing step is accomplished bytangential flow filtration (TFF) according to the manufacturer'sinstructions. In some embodiments, the cells are resuspended in avariety of biocompatible buffers after washing, such as, for example,Ca⁺⁺/Mg⁺⁺ free PBS. In certain embodiments, components of a blood cellsample are removed and the cells directly resuspended in culture media.

In some embodiments, the methods include density-based cell separationmethods, such as the preparation of white blood cells from peripheralblood by lysing the red blood cells and centrifugation through a Percollor Ficoll gradient.

4. Separation Based on Affinity and/or Marker Profile

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population.

The separation need not result in 100% enrichment or removal of aparticular cell population or cells expressing a particular marker. Forexample, positive selection of or enrichment for cells of a particulartype, such as those expressing a marker, refers to increasing the numberor percentage of such cells, but need not result in a complete absenceof cells not expressing the marker. Likewise, negative selection,removal, or depletion of cells of a particular type, such as thoseexpressing a marker, refers to decreasing the number or percentage ofsuch cells, but need not result in a complete removal of all such cells.For example, in some aspects, a selection of one of the CD4+ or CD8+population enriches for said population, either the CD4+ or CD8+population, but also can contain some residual or small percentage ofother non-selected cells, which can, in some cases, include the other ofthe CD4 or CD8 population still being present in the enrichedpopulation.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28⁺, CD62L⁺, CCR7⁺, CD27⁺, CD127⁺, CD4⁺, CD8⁺, CD45RA⁺,and/or CD45RO⁺ T cells, are isolated by positive or negative selectiontechniques.

For example, CD3⁺, CD28⁺ T cells can be positively selected usingCD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 TCell Expander).

In some embodiments, isolation is carried out by enrichment for aparticular cell population by positive selection, or depletion of aparticular cell population, by negative selection. In some embodiments,positive or negative selection is accomplished by incubating cells withone or more antibodies or other binding agent that specifically bind toone or more surface markers expressed or expressed (marker) at arelatively higher level (marker^(high)) on the positively or negativelyselected cells, respectively.

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In someembodiments, the methods include isolation, selection and/or enrichmentof CD4+ and CD8+ cells. In one example, to enrich for CD4⁺ cells bynegative selection, a monoclonal antibody cocktail typically includesantibodies to CD14, CD20, CD11b, CD16 and HLA-DR. In one example, toenrich for a CD8⁺ population by negative selection is carried out bydepletion of cells expressing CD14 and/or CD45RA. In some aspects, aCD4⁺ or CD8⁺ selection step, such as positive selection for CD4 andpositive selection for CD8, is used to separate CD4⁺ helper and CD8⁺cytotoxic T cells. Such selections in some aspects are carried outsimultaneously and in other aspects are carried out sequentially, ineither order.

In some aspects, the methods include a first positive selection for CD4⁺cells in which the non-selected cells (CD4− cells) from the firstselection are used as the source of cells for a second positiveselection to enrich for CD8+ cells. In some aspects, the methods includea first positive selection for CD8+ cells in which the non-selectedcells (CD8− cells) from the first selection are used as the source ofcells for a second position selection to enrich for CD4+ cells. SuchCD4⁺ and CD8⁺ populations can be further sorted into sub-populations bypositive or negative selection for markers expressed or expressed to arelatively higher degree on one or more naive, memory, and/or effector Tcell subpopulations.

In some embodiments, CD4+ cells are further enriched for or depleted ofnave, central memory, effector memory and/or central memory stem cells,such as by positive or negative selection based on surface antigensassociated with the respective population. CD4⁺ T helper cells aresorted into nave, central memory, and effector cells by identifying cellpopulations that have cell surface antigens. CD4⁺ lymphocytes can beobtained by standard methods. In some embodiments, naive CD4⁺ Tlymphocytes are CD45RO⁻, CD45RA⁺, CD62L⁺, CD4⁺ T cells. In someembodiments, central memory CD4⁺ cells are CD62L⁺ and CD45RO⁺. In someembodiments, effector CD4⁺ cells are CD62L⁻ and CD45RO

In some embodiments, CD8⁺ cells are further enriched for or depleted ofnaive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (T_(CM)) cells is carriedout to increase efficacy, such as to improve long-term survival,expansion, and/or engraftment following administration, which in someaspects is particularly robust in such sub-populations. See Terakura etal. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother.35(9):689-701. In some embodiments, combining T_(CM)-enriched CD8⁺ Tcells and CD4⁺ T cells further enhances efficacy.

In embodiments, memory T cells are present in both CD62L⁺ and CD62L−subsets of CD8⁺ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L-CD8⁺ and/or CD62L⁺CD8⁺ fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (T_(CM)) cellsis based on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, CD27 and/or CD 127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B.

In some embodiments, the provided methods include isolation, selectionand/or enrichment of CD8+ cells from a sample, such as by positiveselection based on surface expression of CD8. In some embodiments, themethods can further include enriching for central memory T (T_(CM))cells. In one aspect, the enriched CD8+ cells can be further enrichedfor central memory T (T_(CM)) cells by selecting for one or more markersexpressed on central memory T (T_(CM)) cells, such as one or more ofCD45RO, CD62L, CCR7, CD28, CD3, CD27 and/or CD 127. The selection can beperformed prior to or subsequent to isolation, selection and/orenrichment of CD4+ cells. Such selections in some aspects are carriedout simultaneously and in other aspects are carried out sequentially, ineither order.

In some aspects, the methods include a first positive selection for CD4+cells in which the non-selected cells (CD4− cells) from the firstselection are used as the source of cells for a second selection toenrich for CD8+ cells, and the enriched or selected CD8+ cells are usedin a third selection to further enrich for cells expressing one or moremarkers expressed on central memory T (T_(CM)) cells, such as by a thirdselection to enrich for CD45RO+, CD62L+, CCR7+, CD28+, CD3+, CD27+and/or CD127+ cells. In some aspects, the methods include a firstpositive selection for CD8+ cells in which the non-selected cells (CD8−cells) from the first selection are used as the source of cells for thesecond selection to enrich for CD4+ cells, and the enriched or selectedCD8+ cells from the first selection also are used in a third selectionto further enrich for cells expressing one or more markers expressed oncentral memory T (T_(CM)) cells, such as by a third selection to enrichfor CD45RO+, CD62L+, CCR7+, CD28+, CD3+, CD27+ and/or CD127+ cells.

In some aspects, isolation of a CD8⁺ population enriched for T_(CM)cells is carried out by depletion of cells expressing CD4, CD14, CD45RA,and positive selection or enrichment for cells expressing CD62L. In oneaspect, enrichment for central memory T (T_(CM)) cells is carried outstarting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8⁺ cell population or subpopulation, also is used to generate theCD4⁺ cell population or sub-population, such that both the positive andnegative fractions from the CD4− based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4⁺ cells, where both the negativeand positive fractions are retained. The negative fraction then issubjected to negative selection based on expression of CD14 and CD45RAor CD19, and positive selection based on a marker characteristic ofcentral memory T cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

In some embodiments, the methods of isolating, selecting and/orenriching for cells, such as by positive or negative selection based onthe expression of a cell surface marker or markers, for example by anyof the methods described above, can include immunoaffinity-basedselections. In some embodiments, the immunoaffinity-based selectionsinclude contacting a sample containing cells, such as primary human Tcells containing CD4+ and CD8+ cells, with an antibody or bindingpartner that specifically binds to the cell surface marker or markers.In some embodiments, the antibody or binding partner is bound to a solidsupport or matrix, such as a sphere or bead, for example microbeads,nanobeads, including agarose, magnetic bead or paramagnetic beads, toallow for separation of cells for positive and/or negative selection. Insome embodiments, the spheres or beads can be packed into a column toeffect immunoaffinity chromatography, in which a sample containingcells, such as primary human T cells containing CD4+ and CD8+ cells, iscontacted with the matrix of the column and subsequently eluted orreleased therefrom.

a. Immunoaffinay Beads

For example, in some embodiments, the cells and cell populations areseparated or isolated using immunomagnetic (or affinitymagnetic)separation techniques (reviewed in Methods in Molecular Medicine, vol.58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and InVivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana PressInc., Totowa, N.J.).

In some aspects, the sample or composition of cells to be separated isincubated with small, magnetizable or magnetically responsive material,such as magnetically responsive particles or microparticles, such asparamagnetic beads. The magnetically responsive material, e.g.,particle, generally is directly or indirectly attached to a bindingpartner, e.g., an antibody, that specifically binds to a molecule, e.g.,surface marker, present on the cell, cells, or population of cells thatit is desired to separate, e.g., that it is desired to negatively orpositively select. Such beads are known and are commercially availablefrom a variety of sources including, in some aspects, Dynabeads® (LifeTechnologies, Carlsbad, Calif.), MACS® beads (Miltenyi Biotec, SanDiego, Calif.) or Streptamer® bead reagents (IBA, Germany).

In some embodiments, the magnetic particle or bead comprises amagnetically responsive material bound to a specific binding member,such as an antibody or other binding partner. There are many well-knownmagnetically responsive materials used in magnetic separation methods.Suitable magnetic particles include those described in Molday, U.S. Pat.No. 4,452,773, and in European Patent Specification EP 452342 B, whichare hereby incorporated by reference. Colloidal sized particles, such asthose described in Owen U.S. Pat. No. 4,795,698, and Liberti et al.,U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby theantibodies or binding partners, or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In some aspects, the sample is placed in a magnetic field, and thosecells having magnetically responsive or magnetizable particles attachedthereto will be attracted to the magnet and separated from the unlabeledcells. For positive selection, cells that are attracted to the magnetare retained; for negative selection, cells that are not attracted(unlabeled cells) are retained. In some aspects, a combination ofpositive and negative selection is performed during the same selectionstep, where the positive and negative fractions are retained and furtherprocessed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody- or otherbinding partner (e.g., streptavidin)-coated magnetic particles, areadded. In certain embodiments, streptavidin-coated magnetic particlesare used in conjunction with biotinylated primary or secondaryantibodies.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies,magnetizable particles or antibodies conjugated to cleavable linkers,etc. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is viamagnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn,Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable ofhigh-purity selection of cells having magnetized particles attachedthereto. In certain embodiments, MACS operates in a mode wherein thenon-target and target species are sequentially eluted after theapplication of the external magnetic field. That is, the cells attachedto magnetized particles are held in place while the unattached speciesare eluted. Then, after this first elution step is completed, thespecies that were trapped in the magnetic field and were prevented frombeing eluted are freed in some manner such that they can be eluted andrecovered. In certain embodiments, the non-target cells are labelled anddepleted from the heterogeneous population of cells.

In some embodiments, the affinity-based selection employs Streptamers®,which are magnetic beads, such as nanobeads or microbeads, for example1-2 μM that, in some aspects, are conjugated to a binding partnerimmunoaffinity reagent, such as an antibody via a streptavidin mutant,e.g. Strep-Tactin® or Strep-Tactin XT® (see e.g. U.S. Pat. No.6,103,493, International Published PCT Appl. Nos. WO/2013011011, WO2014/076277). In some embodiments, the streptavidin mutant isfunctionalized, coated and/or immobilized on the bead.

In some embodiments, the streptavidin mutant exhibits a higher bindingaffinity for a peptide ligand containing the sequence of amino acids setforth in any of SEQ ID NOS:1-6, such as for example SEQ ID NO:5 and/orSEQ ID NO:6 (e.g. Strep-tag II®), than an unmodified or wild typestreptavidin, such as an unmodified or wild type streptavidin set forthin SEQ ID NO: 11 or SEQ ID NO:14. In some embodiments, the streptavidinmutant exhibits a binding affinity as an affinity constant for suchpeptides that is greater than the binding affinity of wild typestreptavidin for the same peptide by greater than 5-fold, 10-fold,50-fold, 100-fold, 200-fold or greater.

The streptavidin mutein contains one or more amino acid differencescompared to an unmodified streptavidin, such as a wild type streptavidinor fragment thereof. The term “unmodified streptavidin” refers to astarting polypeptide to which one or more modifications are made. Insome embodiments, the starting or unmodified polypeptide may be a wildtype polypeptide set forth in SEQ ID NO:11. In some embodiments, theunmodified streptavidin is a fragment of wild type streptavidin, whichis shortened at the N- and/or C-terminus. Such minimal streptavidinsinclude any that begin N-terminally in the region of amino acidpositions 10 to 16 of SEQ ID NO:11 and terminate C-terminally in theregion of amino acid positions 133 to 142 of SEQ ID NO:11. In someembodiments, the unmodified streptavidin has the sequence of amino acidsset forth in SEQ ID NO:14. In some embodiments, the unmodifiedstreptavidin, such as set forth in SEQ ID NO:14, can further contain anN-terminal methionine at a position corresponding to Ala13 withnumbering as set forth in SEQ ID NO:11. Reference to number of residuesin streptavidin provided herein is with reference to numbering ofresidues in SEQ ID NO:11.

The term “streptavidin mutein,” “streptavidin mutant” or variationsthereof, refers to a streptavidin protein that contains one or moreamino acid differences compared to an unmodified or wild typestreptavidin, such as a streptavidin set forth in SEQ ID NO: 11 or SEQID NO:14. The one or more amino acid differences can be amino acidmutations, such as one or more amino acid replacements (substitutions),insertions or deletions. In some embodiments, a streptavidin mutein canhave at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 amino acid differences compared to a wild type orunmodified streptavidin. In some embodiments, the amino acidreplacements (substitutions) are conservative or non-conservativemutations. The streptavidin mutein containing the one or more amino aciddifferences exhibits a binding affinity as an affinity constant that isgreater than 2.7×10⁴ M⁻¹ for the peptide ligand (Trp Arg His Pro Gln PheGly Gly; also called Strep-Tag®, set forth in SEQ ID NO:5). In someembodiments, the streptavidin mutant exhibits a binding affinity as anaffinity constant that is greater than 1.4×10⁴ M⁻¹ for the peptideligand (Trp Ser His Pro Gln Phe Glu Lys; also called Strep-Tag® II, setforth in SEQ ID NO:6). In some embodiments, binding affinity can bedetermined by methods known in the art, such as any described below.

In some embodiments, the streptavidin mutein contains a mutation at oneor more residues 44, 45, 46, and/or 47. In some embodiments, thestreptavidin mutant contains residues Val44-Thr45-Ala46-Arg47, such asset forth in exemplary streptavidin muteins set forth in SEQ ID NO: 12or SEQ ID NO:15. In some embodiments, the streptavidin mutein containsresidues Ile44-Gly45-Ala-46-Arg47, such as set forth in exemplarystreptavidin muteins set forth in SEQ ID NO: 13 or 16. In someembodiments, the streptavidin mutein exhibits the sequence of aminoacids set forth in SEQ ID NO: 12, 13, 15 or 16, or a sequence of aminoacids that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to the sequence of amino acids setforth in SEQ ID NO:12, 13, 15 or 16, and exhibits a binding affinitythat is greater than 2.7×10⁴ M⁻¹ for the peptide ligand (Trp Arg His ProGln Phe Gly Gly; also called Strep-Tag®, set forth in SEQ ID NO:5)and/or greater than 1.4×10⁴ M⁻¹ for the peptide ligand (Trp Ser His ProGln Phe Glu Lys; also called Strep-Tag® II, set forth in SEQ ID NO:6).

In some embodiment, the streptavidin mutein is a mutant as described inInternational Published PCT Appl. Nos. WO 2014/076277. In someembodiments, the streptavidin mutein contains at least two cysteineresidues in the region of amino acid positions 44 to 53 with referenceto amino acid positions set forth in SEQ ID NO:11. In some embodiments,the cysteine residues are present at positions 45 and 52 to create adisulfide bridge connecting these amino acids. In such an embodiment,amino acid 44 is typically glycine or alanine and amino acid 46 istypically alanine or glycine and amino acid 47 is typically arginine. Insome embodiments, the streptavidin mutein contains at least one mutationor amino acid difference in the region of amino acids residues 115 to121 with reference to amino acid positions set forth in SEQ ID NO:11. Insome embodiments, the streptavidin mutein contains at least one mutationat amino acid position 117, 120 and 121 and/or a deletion of amino acids118 and 119 and substitution of at least amino acid position 121.

In some embodiments, a streptavidin mutein can contain any of the abovemutations in any combination, so long as the resulting streptavidinmutein exhibits a binding affinity that is greater than 2.7×10⁴ M⁻¹ forthe peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also calledStrep-Tag®, set forth in SEQ ID NO:5) and/or greater than 1.4×10⁴ M⁻¹for the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also calledStrep-Tag® II, set forth in SEQ ID NO:6).

In some embodiments, the binding affinity of a streptavidin mutant for apeptide ligand binding reagent is greater than 5×10⁴ M⁻¹, 1×10⁵ M⁻¹,5×10⁵ M⁻¹, 1×10⁶ M⁻¹, 5×10⁶ M⁻¹ or 1×10⁷ M⁻¹, but generally is less than1×10¹³ M⁻¹, 1×10¹² M⁻¹ or 1×10¹¹ M.

In some embodiments, the streptavidin mutant also exhibits binding toother streptavidin ligands, such as but not limited to, biotin,iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA(hydroxyazobenzene-benzoic acid) or/and dimethyl-HABA. In someembodiments, the streptavidin muteins exhibits a binding affinity foranother streptavidin ligand, such as biotin or desthiobiotin, that isgreater than the binding affinity of the streptavidin mutein for thepeptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called Strep-Tag®,set forth in SEQ ID NO:5) or the peptide ligand (Trp Ser His Pro Gln PheGlu Lys; also called Strep-Tag® II, set forth in SEQ ID NO:6).

In some embodiments, the streptavidin mutein is a multimer. Multimerscan be generated using any methods known in the art, such as anydescribed in published U.S. Patent Application No. US2004/0082012. Insome embodiments, oligomers or polymers of muteins can be prepared bythe introduction of carboxyl residues into a polysaccharide, e.g.dextran. In some aspects, streptavidin muteins then are coupled viaprimary amino groups of internal lysine residues and/or the freeN-terminus to the carboxyl groups in the dextran backbone usingconventional carbodiimide chemistry in a second step. In someembodiments, the coupling reaction is performed at a molar ratio ofabout 60 moles streptavidin mutant per mole of dextran. In someembodiments, oligomers or polymers of can also be obtained bycrosslinking via bifunctional linkers, such as glutardialdehyde or byother methods known in the art.

In some aspects an immunoaffinity bead, such as a Streptamer® or otherimmunoaffinity bead, can contain an antibody produced by or derived froma hybridoma as follows: OKT3 (αCD3), 13B8.2 (αCD4), OKT8 (αCD8), FRT5(αCD25), DREG56 (αCD62L), MEM56 (αCD45RA). In some embodiments, any ofthe above antibodies can contain one or more mutations within theframework of heavy and light chain variable regions without targetingthe highly variable CDR regions. Exemplary of such antibodies include,in some aspects, anti-CD4 antibodies as described in U.S. Pat. No.7,482,000 and Bes et al. (2003) J. Biol. Chem., 278:14265-14273. In someembodiments, an antigen-binding fragment, such as a Fab fragment, can begenerated from such antibodies using methods known in the art, such as,in some aspects, amplification of hypervariable sequences of heavy andlight chains and cloning to allow combination with sequences coding foran appropriate constant domain. In some embodiments, the constant domainis of human subclass IgG 1/κ. Such antibodies can be carboxy-terminallyfused with a peptide streptavidin binding molecule, such as set forth inSEQ ID NO:10. Exemplary of such antibodies are described in Stembergetet al. (2102) PLoS One, 7:35798 and International PCT Application No.WO2013/011011.

In some embodiments, the antibody specifically binding a cell surfacemarker associated with or coated on a bead or other surface is afull-length antibody or is an antigen-binding fragment thereof,including a (Fab) fragments, F(ab′)₂ fragments, Fab′ fragments, Fvfragments, variable heavy chain (V_(H)) regions capable of specificallybinding the antigen, single chain antibody fragments, including singlechain variable fragments (scFv), and single domain antibodies (e.g.,sdAb, sdFv, nanobody) fragments. In some embodiments, the antibody is aFab fragment. In some embodiments, the antibody can be monovalent,bivalent or multivalent. In some embodiments, the antibody, such as aFab, is a multimer. In some embodiments, the antibody, such as a Fabmultimer, forms a multivalent complex with the cell surface marker.

In some embodiments, the antibody, such as a Fab, associated with aStreptamer exhibits a particular kinetic measure of binding affinity(e.g. dissociation constant, K_(D), association constant K_(A), off-rateor other kinetic parameter of binding affinity). Such measurements canbe determined using any binding assay known to a skilled artisan. Inparticular examples, an affinity-based biosensor technology is utilizedas a measure of binding affinity. Exemplary biosensor technologiesinclude, for example, Biacore technologies, BioRad ProteOn, Reichert,GWC Technologies, IBIS SPIR Imaging, Nomadics SensiQ, Akubio RAPid,ForteBio Octet, IAsys, Nanofilm and others (see e.g. Rich et al. (2009)Analytical Biochemistry, 386:194-216). In some embodiments, bindingaffinity is determined by fluorescence titration or titrationcalorimetry.

In some embodiments, the antibody, such as a Fab, exhibits a k_(0ff)rate (also called dissociation rate constant) for the binding to a cellsurface marker on a cell that is greater than about 0.5×10⁻⁴ sec⁻¹,about 1×10⁻⁴ sec⁻¹, about 2×10⁻⁴ sec⁻¹, about 3×10⁻⁴ sec⁻¹, about 4×10⁻⁴sec⁻¹, about 5×10⁻⁴ sec⁻¹, about 1×10⁻³ sec⁻¹, about 1.5×10⁻³ sec⁻¹,about 2×10⁻³ sec⁻¹, about 3×10⁻³ sec⁻¹, about 4×10⁻³ sec⁻¹, about 5×10⁻³sec⁻¹, about 1×10⁻² sec⁻¹, or about 5×10⁻¹ sec⁻¹ or greater. Theparticular k_(0ff) rate can determine the rate at which the antibodyreagent can dissociate from the its interaction with a cell via itsbinding to a cell surface marker (see e.g. International Published PCTAppl. No. WO/2013011011). For example, in some aspects, the k_(0ff)range can be chosen within a range, depending, for example, on theparticular application or use of a selected or enriched cell, includingfactors such as the desire to remove the bound antibody from the cellsurface, the time of the cells in culture or incubation, the sensitivityto the cell and other factors. In one embodiment, an antibody has a highk_(0ff) rate of, for example, greater than 4.0×10⁻⁴ sec⁻¹, so that,after the disruption of the multivalent binding complexes, most of theantibody can be removed within one hour, since, in some aspects takinginto account the half-life T½ of the complex, within 56 minutes theconcentration of the complexes is reduced to 25% of the originalconcentration, assuming that rebinding effects can be neglected due tosufficient dilution). In another embodiment, an antibody with a lowerk0ff rate of, for example, 1.0×10⁻⁴ sec⁻¹, the dissociation may takelonger, for example about or approximately 212 min or about 3 and a halfhours to remove 75% of the antibody from the surface.

In some embodiments, the antibody, such as a Fab, exhibits adissociation constant (Ka) for the binding to a cell surface marker on acell that can be the range of about 10⁻² M to about 10⁻⁸ M, or of about10⁻² M to about 10⁻⁹ M, or of about 10⁻² M to about 0.8×10⁻⁹ M, or ofabout 10⁻² M to about 0.6×10⁻⁹ M, or of about 10⁻² M to about 0.4×10⁻⁹M, or of about 10⁻² M to about 0.3×10⁻⁹ M, or of about 10⁻² M to about0.2×10⁻⁹, or of about 10⁻² M to about 0.15×10⁻⁹ M, or of about 10⁻² toabout 10⁻¹⁰, In some embodiments, the dissociation constant (Ka) for thebinding a cell surface marker on a cell can be in the range of about10⁻⁷ M to about 10⁻¹⁰ M, or of about 10⁻⁷ M to about 0.8×10⁻⁹ M, or ofabout 10⁻⁷ M to about 0.6×10⁻⁹ M, of about 10⁻⁷ M to about 0.3×10⁻⁹ M,of 1.1×10⁻⁷ M to about 10⁻¹⁰ M, or of about 1.1×10⁻⁷ M to about0.15×10⁻⁹ M, or of about 1.1×10⁻⁷ M to about 0.3×10⁻⁹ M, or of about1.1×10⁻⁷ M to about 0.6×10⁻⁹ M, or of about 1.1×10⁻⁷ M to about 0.8×10⁹M.

In some embodiments, the immunoaffinity reagent, such as antibody, forexample a Fab, is linked, directly or indirectly, to a peptide ligand,such as a peptide ligand capable of binding to a streptavidin mutant(see e.g. U.S. Pat. No. 5,506,121). In some embodiments, such as peptidecontains the sequence of amino acids set forth in any of SEQ ID NOS:1-6.In some embodiments, the immunoaffinity reagent, such as antibody, forexample a Fab, is linked, directly or indirectly, to a peptide ligandcontaining the sequence of amino acids set forth in SEQ ID NO:6.

In some embodiments, the immunoaffinity reagent, such as antibody, forexample a Fab, is fused directly or indirectly with a peptide sequencethat contains a sequential arrangement of at least twostreptavidin-binding modules, wherein the distance between the twomodules is at least 0 and not greater than 50 amino acids, wherein onebinding module has 3 to 8 amino acids and contains at least the sequenceHis-Pro-Xaa (SEQ ID NO:1), where Xaa is glutamine, asparagine, ormethionine, and wherein the other binding module has the sequence of thesame or different streptavidin peptide ligand, such as set forth in SEQID NO:3 (see e.g. International Published PCT Appl. No. WO02/077018;U.S. Pat. No. 7,981,632). In some embodiments, the peptide ligand fused,directly or indirectly, to the immunoaffinity reagent, such as antibody,for example Fab, contains a sequence having the formula set forth in anyof SEQ ID NO: 7 or 8. In some embodiments, the peptide ligand has thesequence of amino acids set forth in any of SEQ ID NOS: 9, 10 or 17-19.

Alternatively, other streptavidin-binding peptides known in the art maybe used, e.g. as described by Wilson et al. (Proc. Natl. Acad. Sci. USA98 (2001), 3750-3755). In some embodiments, the peptide is fused to theN- and/or C-terminus of the protein.

In some embodiments, the antibody, such as a Fab, fused to a peptideligand capable of binding a streptavidin mutant, is contacted withstreptavidin-mutant containing beads to coat the beads with antibody. Insome embodiments, the coated beads can be used in enrichment andselection methods as described herein by contacting such beads with asample containing cells to be enriched or selected.

In some embodiments, the bond between the peptide ligand binding partnerand streptavidin mutein binding reagent is reversible. In someembodiments, the bond between the peptide ligand binding partner andstreptavidin mutein binding reagent is high, such as described above,but is less than the binding affinity of the streptavidin bindingreagent for biotin or a biotin analog. Hence, in some embodiments,biotin (Vitamin H) or a biotin analog can be added to compete forbinding to disrupt the binding interaction between the streptavidinmutein binding reagent on the bead and the peptide ligand bindingpartner associated with the antibody specifically bound to a cell markeron the surface. In some embodiments, the interaction can be reversed inthe presence of low concentrations of biotin or analog, such as in thepresence of 0.1 mM to 10 mM, 0.5 mM to 5 mM or 1 mM to 3 mM, such asgenerally at least or about at least 1 mM or at least 2 mM, for exampleat or about 2.5 mM. In some embodiments, incubation in the presence of acompeting agent, such as a biotin or biotin analog, releases the beadfrom the selected cell.

b. Immunoaffinity Chromatography

In some embodiments, the affinity-based selection employs immunoaffinitychromatography. Immunoaffinity chromatography methods include, in someaspects, one or more chromatography matrix as described in U.S.Published Patent Appl. No. US2015/0024411. In some embodiments, thechromatographic method is a fluid chromatography, typically a liquidchromatography. In some embodiments, the chromatography can be carriedout in a flow through mode in which a fluid sample containing the cellsto be isolated is applied, for example, by gravity flow or by a pump onone end of a column containing the chromatography matrix and in whichthe fluid sample exits the column at the other end of the column. Inaddition, in some aspects, the chromatography can be carried out in an“up and down” mode in which a fluid sample containing the cells to beisolated is applied, for example, by a pipette on one end of a columncontaining the chromatography matrix packed within a pipette tip and inwhich the fluid sample enters and exits the chromatographymatrix/pipette tip at the other end of the column. In some embodiments,the chromatography can also be carried out in a batch mode in which thechromatography material (stationary phase) is incubated with the samplethat contains the cells, for example, under shaking, rotating orrepeated contacting and removal of the fluid sample, for example, bymeans of a pipette.

In some embodiments, the chromatography matrix is a stationary phase. Insome embodiments, the chromatography is column chromatography. In someembodiments, any suitable chromatography material can be used. In someembodiments, the chromatography matrix has the form of a solid orsemi-solid phase. In some embodiments, the chromatography matrix caninclude a polymeric resin or a metal oxide or a metalloid oxide. In someembodiments, the chromatography matrix is a non-magnetic material ornon-magnetizable material. In some embodiments, the chromatographymatrix is a derivatized silica or a crosslinked gel, such as in the formof a natural polymer, for example a polysaccharide. In some embodiments,the chromatography matrix is an agarose gel. Agarose gel for use in achromatography matrix are known in the art and include, in some aspects,Superflow™ agarose or a Sepharose material such as Superflow™Sepharose®, which are commercially available in different bead and poresizes. In some embodiments, the chromatography matrix is a particularcross-linked agarose matrix to which dextran is covalently bonded, suchas any known in the art, for example in some aspects, Sephadex®,Superdex® or Sephacryl®, which are available in different bead and poresizes.

In some embodiments, a chromatography matrix is made of a syntheticpolymer, such as polyacrylamide, a styrene-divinylbenzene gel, acopolymer of an acrylate and a diol or of an acrylamide and a diol, aco-polymer of a polysaccharide and agarose, e.g. apolyacrylamide/agarose composite, a polysaccharide andN,N′-methylenebiscarylamide, or a derivatized silica coupled to asynthetic or natural polymer.

In some embodiments, the chromatography matrix, such as agarose beads orother matrix, has a size of at least or about at least 50 μm, 60 μm, 70μm, 80 μm, 90 μm, 100 μm, 120 μm or 150 μm or more. The exclusion limitof the size exclusion chromatography matrix is selected to be below themaximal width of the target cell in a sample, e.g. T cells. In someembodiments, the volume of the matrix is at least 0.5 mL, 1 mL, 1.5 mL,2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL or more. In someembodiments, the chromatography matrix is packed into a column.

In some embodiments, the chromatography matrix, which is animmunoaffinity chromatography matrix, includes an affinity reagent, suchas an antibody or antigen-binding fragment, such as Fab, immobilizedthereto. The antibody or antigen-binding fragment, such as a Fab, can beany as described above, including, in some aspects, known antibodies inthe art, antibodies having a particular k_(0ff) rate and/or antibodieshaving a particular dissociation constant (Ka).

In some embodiments, the affinity reagent, such as antibody orantigen-binding fragment, such as a Fab, is immobilized. In someembodiments, the immunoaffinity reagent, such as an antibody orantigen-binding fragment, such as a Fab, is fused or linked to a bindingpartner that interacts with a binding reagent immobilized on the matrix.In some embodiments, the binding capacity of the chromatography matrixis sufficient to adsorb or is capable of adsorbing at least 1×107cells/mL, 5×107 cells/mL, 1×108 cells/mL, 5×108 cells/mL, 1×109 cells/mlor more, in which said cells are cells expressing a cell surface markerspecifically recognized by the affinity reagent, such as antibody orFab.

In some embodiments, the interaction between the binding reagent andbinding partner forms a reversible bond, so that binding of the antibodyto the matrix is reversible. In some embodiments, the reversible bindingcan be mediated by a streptavidin mutant binding partner and a bindingreagent immobilized on the matrix that is streptavidin, a streptavidinanalog or mutein, avidin or an avidin analog or mutein.

In some embodiments, reversible binding of the affinity reagent, such asantibody or antigen-binding fragment, such as Fab is via a peptideligand binding reagent and streptavidin mutein interaction, as describedabove with respect to immunoaffinity beads. In aspects of thechromatography matrix, the matrix, such as agarose beads or othermatrix, is functionalized or conjugated with a streptavidin mutein, suchas any described above, for example any set forth in SEQ ID NOS: 12, 13,15 or 16. In some embodiments, the antibody or antigen-binding fragment,such as a Fab, is fused or linked, directly or indirectly, to a peptideligand capable of binding to a streptavidin mutant, such as anydescribed above. In some embodiments, the peptide ligand is any asdescribed above, such as a peptide containing the sequence of aminoacids set forth in any of SEQ ID NOS:1-10 or 17-19. In some embodiments,the chromatography matrix column is contacted with such an affinityreagent, such as an antibody or antigen-binding fragment, such as a Fabto immobilize or reversibly bind the affinity reagent to the column.

In some embodiments, the immunoaffinity chromatography matrix can beused in enrichment and selection methods as described herein bycontacting said matrix with a sample containing cells to be enriched orselected. In some embodiments, the selected cells are eluted or releasedfrom the matrix by disrupting the interaction of the bindingpartner/binding reagent. In some embodiments, binding partner/bindingreagents is mediated by a peptide ligand and streptavidin mutantinteraction, and the release or selected cells can be effected due tothe presence of a reversible bond. For example, in some embodiments, thebond between the peptide ligand binding partner and streptavidin muteinbinding reagent is high, such as described above, but is less than thebinding affinity of the streptavidin binding reagent for biotin or abiotin analog. Hence, in some embodiments, biotin (Vitamin H) or abiotin analog can be added to compete for binding to disrupt the bindinginteraction between the streptavidin mutein binding reagent on thematrix and the peptide ligand binding partner associated with theantibody specifically bound to a cell marker on the surface. In someembodiments, the interaction can be reversed in the presence of lowconcentrations of biotin or analog, such as in the presence of 0.1 mM to10 mM, 0.5 mM to 5 mM or 1 mM to 3 mM, such as generally at least orabout at least 1 mM or at least 2 mM, for example at or about 2.5 mM. Insome embodiments, elution in the presence of a competing agent, such asa biotin or biotin analog, releases the selected cell from the matrix.

In some embodiments, immunoaffinity chromatography in the providedmethods is performed using at least two chromatography matrix columnsthat are operably connected, whereby an affinity or binding agent to oneof CD4 or CD8, such as an antibody, e.g. a Fab, is coupled to a firstchromatography matrix in a first selection column and an affinity orbinding agent to the other of CD4 or CD8, such as an antibody, e.g. aFab, is coupled to a second chromatography matrix in a second selectioncolumn. In some embodiments, the at least two chromatography matrixcolumns are present in a closed system or apparatus, such as a closedsystem or apparatus that is sterile.

In some embodiments, also provided herein is a closed system orapparatus containing at least two chromatography matrix columns that areoperably connected, whereby an affinity or binding agent to one of CD4or CD8, such as an antibody, e.g. a Fab, is coupled to a firstchromatography matrix in a first selection column and an affinity orbinding agent to the other of CD4 or CD8, such as an antibody, e.g. aFab, is coupled to a second chromatography matrix in a second selectioncolumn. Exemplary of such systems and methods are depicted in FIG. 1A anFIG. 1B, and in Examples.

In some embodiments, the closed system is automated. In someembodiments, components associated with the system can include anintegrated microcomputer, peristaltic pump, and various valves, such aspinch valves or stop cocks, to control flow of fluid between the variousparts of the system. The integrated computer in some aspects controlsall components of the instrument and directs the system to performrepeated procedures in a standardized sequence. In some embodiments, theperistaltic pump controls the flow rate throughout the tubing set and,together with the pinch valves, ensures the controlled flow of bufferthrough the system.

With reference to FIG. 1A and FIG. 1B, in some embodiments, the firstaffinity matrix 3 containing a first affinity or binding agent in afirst selection column 1 is i) operably coupled to the second affinitymatrix 4 containing a second affinity or binding agent in a secondselection column 2 via tubing and a valve 13 so that cells having passedthrough the first affinity chromatography matrix and not being bound toa first affinity or binding agent thereon are capable of being passedinto the second affinity matrix and ii) is operably coupled to an outputcontainer, such as a culture vessel 12, to collect selected cells fromthe first affinity matrix that bound to the first affinity or bindingagent thereon, such as after elution and release of such cells from thefirst affinity matrix. In some embodiments, the second affinity matrix 4containing a second affinity or binding agent in a second selectioncolumn 2 also is operably coupled to the output container, such as aculture vessel 12, to collect selected cells from the second affinitymatrix that bound to the second affinity or binding agent thereon, suchas after elution and release of such cells from the second affinitymatrix. In some embodiments, the second selection column 2 is operablyconnected to the output container, such as culture vessel 12, through aremoval chamber 9 that contains a binding reagent, such as astreptavidin mutant, that is able to bind with high affinity, such asgreater than 10⁻¹⁰ M⁻¹, to an elution reagent, such as biotin.

In some embodiments, the size, e.g. length and/or diameter, of the firstselection column 1 and second selection column 2 can be the same ordifferent. In some embodiments, the size, e.g. length and/or diameter,of one of the first column 1 or second column 2 is larger than the sizeof the other of the columns by at least 1.2 times, 1.5 times, 2 times, 3times, 4 times, 5 times, 5 times, 7 times, 8 times, 9 times, 10 times ormore.

In some embodiments, the first affinity matrix 3 containing a firstaffinity or binding agent in a first selection column 1 also is operablyconnected to a third affinity matrix 17 containing a third affinity orbinding agent in a third selection column 15 via tubing and a valve 13,so that selected cells from the first affinity matrix that bound to thefirst affinity or binding agent thereon are capable of being passed intothe third affinity matrix, such as after elution and release of suchcells from the first affinity matrix. In some embodiments, the firstselection column 1 is operably connected to the third selection column15 through a removal chamber 9 that contains a binding reagent, such asa streptavidin mutant, that is able to bind with high affinity, such asgreater than 10⁻¹⁰ M⁻¹, to an elution reagent, such as biotin. In someembodiments, the third affinity matrix 17 containing a third affinity orbinding agent in a third selection column 15 also is operably coupled tothe output container, such as a culture vessel 12, to collect selectedcells from the third affinity matrix having bound to the third affinityor binding agent thereon (and previously having bound to the firstaffinity or binding agent), such as after elution and release of suchcells from the third affinity matrix (and previously from the firstaffinity matrix). In some embodiments, the third selection column 15 isoperably connected to the output container, such as culture vessel 12,through a removal chamber 9 that contains a binding reagent, such as astreptavidin mutant, that is able to bind with high affinity, such asgreater than 10⁻¹⁰ M¹, to an elution reagent, such as biotin.

In some embodiments, the size, e.g. length and/or diameter, of the firstselection column 1 and third selection column 15 can be the same ordifferent. In some embodiments, the size, e.g. length and/or diameter,of one of the first column 1 or third column 15 is larger than the sizeof the other of the columns by at least 1.2 times, 1.5 times, 2 times, 3times, 4 times, 5 times, 5 times, 7 times, 8 times, 9 times, 10 times ormore.

In some embodiments, the first selection column 1 is operably coupled toa storage reservoir containing cell sample 5, such as via tubing, valvesand a pump 8, in order to provide a cell sample into the first selectioncolumn.

In some embodiments, the first selection column 1 also is operablycoupled to a washing reservoir 6 containing wash buffer and/or anelution reservoir 7 containing an eluent, such as through tubing andvalves, in order to permit passage of a washing buffer or an elutionbuffer, respectively, into the first chromatography matrix in the firstselection column. In some embodiments, due to the operable connectionbetween the first and second selection column, the washing buffer and/orelution buffer can operably pass through into the second column. In someembodiments, due to the operable connection between the first and thirdselection column, the washing buffer and/or elution buffer can operablypass through into the third selection column.

In some embodiments, the second selection column 2 also is operablycoupled to a washing reservoir 6 containing wash buffer and/or anelution reservoir 7 containing an eluent, such as through tubing andvalves, in order to permit passage of a washing buffer or an elutionbuffer, respectively, into the first chromatography matrix in the firstselection column.

The washing buffer can be any physiological buffer that is compatiblewith cells, such as phosphate buffered saline. In some embodiments, thewashing buffer contains bovine serum albumin, human serum albumin, orrecombinant human serum albumin, such as at a concentration of 0.1% to5% or 0.2% to 1%, such as or at about 0.5%. In some embodiments, theeluent is biotin or a biotin analog, such as desbiotin, for example inan amount that is or is about at least 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5mM, 3 mM, 4 mM, or 5 mM.

In some embodiments, the first affinity matrix 3 in the first selectioncolumn 1 is operably connected through tubing and valves to a firstaffinity reagent reservoir (e.g. Fab reservoir) 18 containing the firstaffinity or binding agent, such as an antibody, e.g. a Fab, such as forimmobilization onto the first affinity matrix. In some embodiments, thesecond affinity matrix 4 in the second column 2 is operably connectedthrough tubing and valves to a second affinity or binding agentreservoir (e.g. Fab reservoir) 19 containing the second affinity orbinding agent, such as an antibody, e.g. a Fab, such as forimmobilization onto the second affinity matrix. In some embodiments, thethird affinity matrix 17 in the third selection column 15 is operablyconnected through tubing and valves to a third affinity or binding agentreservoir (e.g. Fab reservoir) 20 containing the third affinity orbinding agent, such as an antibody, e.g. a Fab, such as forimmobilization onto the second affinity matrix.

In some embodiments, the first and/or second affinity reagentspecifically binds CD4 or CD8, where the first and second affinityreagent are not the same. In some embodiments, the third affinityreagent specifically binds a marker on nave, resting or central memory Tcells or specifically binds a marker that is CD45RO, CD62L, CCR7, CD28,CD3, CD27 and/or CD127.

5. Enrichments and Ratios of Generated Compositions

In some embodiments, performing a first and second selection usingmethods as described above enriches from a sample a first population ofcells expressing a first cell surface marker and a second population ofcells expressing a second cell surface marker, respectively. Inparticular examples, the first and/or second population of enrichedcells can be a population of cells enriched for CD4+ cells, and theother of the enriched population of cells, i.e. the other of the firstor second population of cells, can be a population enriched for CD8+. Asdescribed above, in some embodiment, a third, fourth or subsequentselection can be performed to enrich for a further sub-population ofcells from a population of cells previously enriched in the first,second or subsequent enrichments, such as a sub-population of CD4+ cellsand/or a sub-population of CD8+ cells.

In some embodiments, the method produces an enriched composition ofcells containing the first and second population of enriched cells, suchas a population of cells enriched for CD4+ cells and a population ofcells enriched for CD8+ cells. In some embodiments, the enrichedcomposition of cells is designated a culture initiation composition andis used in subsequent processing steps, such as subsequent processingsteps involving incubation, stimulation, activation, engineering and/orformulation of the enriched cells. In some embodiments, subsequent tothe further processing steps, such as processing steps involvingincubation, stimulation, activation, engineering and/or formulation, andoutput composition is generated that, in some aspects, can containgenetically engineered cells containing CD4+ cells and CD8+ cellsexpressing a genetically engineered antigen receptor.

In some embodiments, the enriched compositions of cells are enrichedcells from a starting sample as describe above, in which the number ofcells in the starting sample is at least greater than the desired numberof cells in an enriched composition, such as a culture-initiationcomposition. In some embodiments, the number of cells in the startingsample is greater by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 500%, 1000%, 5000% or more greater than the desired number ofcells in the enriched composition. In some examples, the desired numberof cells in the enriched population, including enriched CD4+ cells, CD8+cells or sub-populations thereof, is at least 1×10⁶ cells, 2×10⁶ cells,4×10⁶ cells 6×10⁶ cells 8×10⁶ cells, 1×10⁷ cells, 2×10⁷ cells, 4×10⁷cells, 6×10⁷ cells, 8×10⁷ cell, 1×10⁸ cells, 2×10⁸ cells, 4×10⁸ cells,6×10⁸ cells, 8×10⁸ cells, 1×10⁹ cells or greater. In some embodiments,the number of cells in the starting sample, is at least 1×10⁸ cells,5×10⁸ cells, 1×10⁹ cells, 2×10⁹ cells, 3×10⁹ cells, 4×10⁹ cells, 5×10⁹cells, 6×10⁹ cells, 7×10⁹ cells, 8×10⁹ cells, 9×10⁹ cells, 1×10¹⁰ cellsor more.

In some embodiments, the yield of the first and/or second population orsub-population thereof, in the enriched composition, i.e. the number ofenriched cells in the population or sub-population compared to thenumber of the same population or sub-population of cells in the startingsample, is 10% to 100%, such as 20% to 80%, 20% to 60%, 20% to 40%, 40%to 80%, 40% to 60%, or 60%, to 80%. In some embodiments, the yield ofthe first and/or second population of cells or sub-population thereof isless than 70%, less than 60%, less than 50%, less than 40%, less than30% or less than 20%.

In some embodiments, the purity of the first and/or second population ofcells or sub-population of cells thereof in the enriched composition,i.e. the percentage of cells positive for the selected cell surfacemarker versus total cells in the population of enriched cells, is atleast 90%, 91%, 92%, 93%, 94%, and is generally at least 95%, 96%, 97%,98%, 99% or greater.

In some embodiments, the enriched composition of cells, such as aculture-initiation composition, contains a ratio of CD4+ cells to CD8+cells at a culture-initiation ratio. The culture-initiation ratio is theratio or number of cells at which two types of cells or isolated cellpopulations are included in a culture-initiating composition, designedto result in the desired output ratio or dose, e.g., ratio or dose foradministration to a patient, or within a tolerated error rate ordifference thereof, at the completion of the incubation and/orengineering step or other processing steps and/or upon thaw and/or justprior to administration to a subject. In embodiments of the methodsprovided herein, the first and/or second selections, or selections forsub-populations thereof, can be performed in a manner to result in achosen culture-initiation ratio. Exemplary of such methods are describedbelow and in Examples.

a. Culture-Initiation Ratios and Numbers

In some embodiments, the culture-initiating ratio of CD4⁺ orsub-populations thereof to CD8⁺ cells or sub-populations thereof isbetween at or about 10:1 and at or about 1:10, between at or about 5:1and at or about 1:5, or between at or about 2:1 and at or about 1:2. Insome embodiments, the culture-initiating ratio of CD4+ cells orsub-populations thereof to CD8+ cells or sub-populations thereof is ator about 1:1.

In some embodiments, the culture-initiating ratio of CD4+ to CD8+ cells,or sub-populations thereof, is different than the ratio of CD4+ to CD8+cells or the sub-populations thereof in the sample from the subject. Insome embodiments, it is reported that the ratio of CD4+ to CD8+ T cellsin a sample from subjects, such as a blood sample, is between 1:1 and14:1 CD4+:CD8+ cells, and generally is between about 1.5:1 and about2.5:1 CD4+:CD8+ cells. In some embodiments, the ratio of CD4+ to CD8+ Tcells in a sample, such as a blood sample, is about 2:1 CD4+:CD8+ cellsIn some embodiments, the ratio of CD4+ to CD8+ T cells in a sample, suchas a blood sample, is about 1:1. (See e.g. Amadori, A et al., NatureMed. 1: 1279-1283, 1995; Chakravarti, A., Nature Med. 1: 1240-1241,1995; Clementi, M., et al., Hum. Genet. 105: 337-342, 1999.) In someembodiments, a subject ratio is less than 1:1 CD4+:CD8+ cells. (See e.g.Muhonen, T. J Immunother Emphasis Tumor Immunol. 1994 January;15(1):67-73). In some embodiments, the culture-initiating ratio of CD4+to CD8+ cells is at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, at least 125%, at least 150, at least 200%, at least 300%,at least 400%, or at least 500% greater or less than the ratio of CD4+to CD8+ cells in the sample from the subject.

In some embodiments, prior to performing the first and/or secondselection, the ratio of CD4+ to CD8+ T cells in the sample from thesubject is determined. Based on the particular ratio of the CD4+ to CD8+T cells in the subject, which can vary among subject, the particularmode of selection can be individualized to the subject, for example bysizing of chromatography columns or selection of amount or concentrationof immunoaffinity reagents, to achieve the desired or chosenculture-initiating ratio. The relative level or frequency of variouscell populations in a subject can be determined based on assessingsurface expression of a marker or markers present on such populations orsub-populations. A number of well-known methods for assessing expressionlevel of surface markers or proteins may be used, such as detection byaffinity-based methods, e.g., immunoaffinity-based methods, e.g., in thecontext of cell surface proteins, such as by flow cytometry.

In some contexts, the appropriate culture-initiating ratio forparticular cell types can vary depending on context, e.g., for example,for a particular disease, condition, or prior treatment of a subjectfrom which cells are derived, and/or a particular antigen-specificity ofthe cells, relative representation among cells of a particular type(e.g., CD8⁺ T cells) of various subpopulations, e.g., effector versusmemory versus nave cells, and/or one or more conditions under whichcells will be incubated, such as medium, stimulating agents, time ofculture, buffers, oxygen content carbon dioxide content, antigen,cytokine, antibodies, and other components. Thus, it may be that a celltype which typically or in general is known to proliferate or expandmore rapidly than another will not always have such a property in everycontext. Thus, in some aspects, the culture-initiation ratio isdetermined based on known capacities of cell types in a normal ortypical context, coupled with assessment of phenotypes or states of thecells or subject from which the cells are derived, and/or empiricalevidence.

In some embodiments, the culture-initiation ratios are based onknowledge of one or more of these features for a particular type of cellknown or determined to be in the concentration. In some embodiments, theratio of CD4⁺ to CD8⁺ cells in the culture-initiating composition isgreater than or less than 1.5 times, 2, times, 3 times, 4 times, 5times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times,40 times, 50 times, 60 times, 70 times, 80 times, 90 times more or less,respectively, than the desired output ratio of CD4⁺ to CD8⁺.

In some embodiments, for example, CD4+ cells in some contexts are knownto proliferate or expand to a lesser degree or less rapidly comparedwith CD8+ cells, when incubated under certain stimulating conditions.See, e.g., Foulds et al. (2002) J Immunol. 168(4): 1528-1532; Caggiariet al. (2001) Cytometry. 46(4) 233-237; Hoffman, et al. (2002)Transplantation. 74(6): 836-845; and Rabenstein et al. (2014) J Immunol.Published online before print Mar. 17, 2014, doi: 10.4049/jimmunol.1302725, Thus, in some examples, the ratio of CD4⁺ to CD8⁺ cells in theculture-initiating composition is 10 times or 100 times the desiredoutput ratio. For example, if a 1:1 (or 50%/50%) CD4/CD8 output ratio isdesired, the CD4⁺ and CD8⁺ populations may in one example be included inthe culture-initiating composition at a ratio of 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1,90:1, or 100:1, for example, to account for differences in expansionrates over a particular period of time. Depending on the differences inrates or expansion or proliferation of one of the CD4+ or CD8+ cells, orsub-populations thereof, from the other, a skilled artisan canempirically determine the culture-initiation ratio to achieve a desiredoutput ratio following under particular conditions of stimulation oractivation.

The culture-initiation ratio will not necessarily be identical, or evenapproximate, the desired output ratio. For example, if it is desired toadminister CD4⁺ and CD8⁺ engineered (e.g., CAR-expressing) T cells at orwithin a certain error of 1:1, the culture-initiation ratio of CD4⁺ andCD8⁺ cells often is not 1:1. In some embodiments, the culture initiationratio that results in the desired output ratio varies depending, e.g.,on the source of the cells, the cell types to be cultured, the patientto whom the cells are to be administered, the subject or subjects fromwhom the cells have been isolated or derived, such as what diseases orconditions such a subject has, the disease to be treated, cultureconditions, and other parameters.

In some embodiments, the culture initiation ratio is based on thecomposition of each subpopulation of cells. In certain embodiments, theculture initiation ratio is based on the length of time the populationsof cells are culture prior to their being genetically engineered. Insome embodiments, the culture initiation ratio is based on theproliferation rate of each population of cells.

As another aspect, in some embodiments, the methods further includedetermining a culture-initiation ratio or number. In some embodiments,the provided methods include methods and steps for determiningappropriate ratios, doses, and numbers of cells, cell types, andpopulations of cells. For example, provided are methods for determiningratios of CD4/CD8⁺ cells, populations, and/or sub-populations anddetermining appropriate doses of such cells and sub-types. In someembodiments, provided are methods for determining appropriate ratios ornumbers of cell types or cell populations to be included in acomposition, such as a culture-initiating composition, to achieve adesired outcome. In some aspects, such ratios or numbers are designedfor use in an incubation or engineering step to achieve a desired outputratio or dose. In some embodiments, provided are methods for determiningdesired ratio of the cells, types, or populations, and/or cell numbersthereof, for administration to a subject or patient.

In some embodiments, the chosen culture initiation ratio is based on therelative capacity of the different cell types or populations forsurvival and/or proliferation or expansion rate of each population ofcells or type of cell (such as CD4⁺ versus CD8⁺ cells) in culture whenincubated under such conditions. Thus, in some aspects, proliferationrate, survival, and/or output ratios are measured or assessed followingtest incubations, for example, at particular point or points in timefollowing incubation, and/or following a cryopreservation or freeze stepand/or following a thaw after such procedure, such as just prior toadministration, e.g., at the bedside, in order to determine the optimalratios for culture initiation. Any of a number of well-known methods fordetermination of in vitro or ex vivo cell proliferation rates orsurvival include flow-cytometry methods such as labeling withcarboxyfluorescein diacetate succinimidyl ester (CFSE) or similarfluorescent dye prior to incubation, followed by assessment offluorescent intensity by flow cytometry, and/or assessment of binding ofcells to Annexin V or other compound recognizing markers on or inapoptotic cells and/or uptake of DNA interchelating agents suchpropidium iodide or 7AAD, and assessment of uptake and/or cell cyclestages as a measure of proliferation or apoptosis by flow cytometry.

In some embodiments, the culture-initiation ratio is determined byincubating two isolated populations, e.g., subpopulations, of cells at arange of different ratios in test compositions, under certain testconditions, and assessing one or more outcomes, such as the output ratioachieved after a certain period. In some aspects, the conditions arestimulatory conditions, such as those approximating conditions underwhich the culture-initiating compositions are to be incubated forculture and/or engineering steps. For example, the test compositions insome aspects are administered in the presence of one or more, e.g.,each, of the same stimulating agents, media, buffers, gas content,and/or in the same type of container or vessel, and/or for the same orapproximately the same amount of time as the parameters to be used inthe incubation and/or engineering steps that are to be used in preparingor producing the ultimate composition, such as the engineeredcomposition for administration.

Exemplary test ratios for the test culture-initiating compositions caninclude at or about 90%/10%, 80%/20%, 70%/30%, 60/40%, 50/50%, 40/60%,30/70%, 20%/80%, and 10%/90%, or 0.1:1, 0.5:1, 0.7:1, 0.8:1, 0.9:1, 1:1,1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or more than at or about 1:1.5, or ator about 1:0.1, 1:0.4, 1:0.7, 1:0.8, 1:0.8, 1.1:1, 1.2:1, 1.3:1, 1.4:1,or 1.5:1, or more.

In some contexts, the appropriate culture-initiation ratio of CD4⁺versus CD8⁺ cells to achieve a desired CD4:CD8 ratio at the end ofproduction is determined based on the sub-population of the CD4⁺ and/orCD8⁺ fractions in a particular isolated cell product, such as thepresence and/or percentage of naive, effector, and various memorycompartments are represented in a particular isolated composition. Suchassessment can be by determining the presence or level of varioussurface markers on the cells, such as by flow cytometry.

In some embodiments, the culture initiation ratio is based on thephenotype of each population of cells. In certain embodiments, theculture initiation ratio is based on the culture conditions (e.g., suchas the composition of the media, the presence and/or absence of growthfactors, stimulants, and/or other agents, temperature, aerationconditions, etc.).

In some embodiments of the methods described herein, the cultureinitiation ratio produces an output composition that comprises a ratioof CD4⁺:CD8⁺ cells or a number of such sub-types or total cell numberthat is within about 10%, about 15%, about 20%, about 25%, about 30%about 35%, about 40%, about 45% or about 50% of the desired ratio ordose, including any range in between these values. In some embodimentsof the methods described herein, the culture initiation ratio producesan output composition having the desired ratio of CD4⁺:CD8⁺ cells ordose of such cells at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more than 95% of the time, including any rangebetween these values. In certain embodiments of the methods, the cultureinitiation ratio produces a ratio of CD4⁺ to CD8⁺ cells in the outputcomposition that is within 20% of the desired output ratio at least 80%of the time. In some embodiments of the methods described herein, theoutput composition comprises a ratio of CD4⁺ to CD8⁺ cells that iswithin a tolerated difference of the desired output ratio, saidtolerated difference having been determined as described above, e.g., byadministering CD4⁺ and CD8⁺ cells to one or more subjects at a pluralityof ratios.

b. Output Ratios and Doses

In some embodiments, the method results an output composition followingone or more processing steps of the culture-initiation composition, suchas incubation, stimulation, activation, engineering and/or formulationof cells. In some embodiments, the method is performed to achieve orresult in a desired ratio of CD4⁺ to CD8⁺ cells, or sub-populationsthereof, that is between at or about 2:1 and at or about 1:5, at orabout 2:1 and at or about 1:2, at or about 1.5:1 and at or about 1:5, ator about 1.5:1 and at or about 1:2, or at or about 1:1 and at or about1:2. In some embodiments, the desired output ratio of CD4⁺ to CD8⁺ cellsin the output composition is 1:1 or is about 1:1.

In some embodiments, the method produces or generates an outputcomposition, such as a composition containing genetically engineeredCD4+ T cells and CD8+ T cells or sub-populations thereof, in which theoutput ratio of CD4+ to CD8+ cells in the composition, orsub-populations thereof, is between at or about 2:1 and at or about 1:5,at or about 2:1 and at or about 1:2, at or about 1.5:1 and at or about1:5, at or about 1.5:1 and at or about 1:2, or at or about 1:1 and at orabout 1:2. In some embodiments, the method produces or generates anoutput composition containing an output ratio of CD4⁺ to CD8⁺ cells thatis 1:1 or is about 1:1.

In some embodiments, the output ratio of CD4+ to CD8+ T cells is a ratiothat is desired as part of a dosage of T cells for immunotherapy, suchas in connection with methods of adoptive immunotherapy.

In some embodiments, desired dosages, such as desired cell numbersand/or desired output ratios of the cell types or populations (e.g.,ratios optimal for therapeutic administration to a patient), aredetermined. In some embodiments, the methods include steps fordetermining tolerated error or tolerated difference from a desiredoutput or administration ratio or dose, i.e., the margin of error bywhich the ratio in a given composition, e.g., engineered composition,can vary from a desired output ratio, and still achieve a desiredoutcome, such as an acceptable degree of safety in a subject or patient,or efficacy in treating a particular disease or condition or othertherapeutic effect.

In some embodiments, the desired dose, ratio and/or the tolerated errordepends on the disease or condition to be treated, subject, source ofcells, such as whether the cells are from a subject having a particularcondition or disease and whether the cells are for autologous orallogeneic transplant, e.g., whether they are isolated from a subjectwho also is to receive the cells in adoptive cell therapy and/or whetherthe subject has received or is receiving another treatment and/or theidentity of such treatment. In some embodiments, the ratio, number,and/or tolerated difference or error depends on one or more otherproperty of the cells, such as proliferation rate, survival capacity,expression of particular markers or secretion of factors, such ascytokines, or particular sub-populations isolated prior to incubationand engineering steps. In some examples, the desired ratio and/or thetolerated error or difference can vary depending on the age, sex,health, and/or weight of the subject, on biomarkers as an indication ofdisease trait, on treatments to be co-administered or having previouslybeen administered to the subject.

In some embodiments, the desired ratio, dose, and/or tolerated error isdetermined by administering various test compositions, each containingthe cell types or populations of interest at different ratios ordifferent numbers to a test subject, followed by assessment of one ormore outcome or parameter, such as a parameter indicative of safety,therapeutic efficacy, in vivo concentration or localization of thecells, and/or other desired outcome.

The test subject in some embodiments is a non-human animal, such as anormal animal or an animal model for disease, such as the disease orcondition to be treated by administration of the cells. In someembodiments, the various test ratios for administration to test subjectsare at or about 90%/10%, 80%/20%, 70%/30%, 60/40%, 50/50%, 40/60%,30/70%, 20%/80%, and 10%/90%, e.g., expressed as percentage of one celltype or sub-type (e.g., CD4⁺ T cell or sub-type thereof) and anothercell type (e.g., CD8⁺ T cell or NK cell or sub-type thereof), andinterim values. The ratios can be expressed as relative percentages orin any other format, such as ratios of at or about 0.1:1, 0.5:1, 0.7:1,0.8:1, 0.9:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or more than at orabout 1:1.5, or at or about 1:0.1, 1:0.4, 1:0.7, 1:0.8, 1:0.8, 1.1:1,1.2:1, 1.3:1, 1.4:1, or 1.5:1, or more, including any range in betweenthese values. In some embodiments, the test subject is a human. In someembodiments, the desired ratio is the average, mean, or median ratioamong test subjects with a particular optimal effect. In some aspects,the desired ratio is a ratio that achieved some optimal balance ofsafety versus efficacy. In some aspects, the desired ratio or dose is aratio or dose that achieves the highest efficacy of all test ratios ordoses, while still maintaining a threshold degree of safety. In someaspects, the desired ratio or dose is a ratio or dose that achieves thehighest degree of safety while maintaining a threshold degree ofefficacy or within a range of efficacy. In some cases, the optimal ratioor dose is expressed as a range, such as between 1:1 and 1:2 of one celltype to another or between 10⁴ and 10⁹ or between 10⁵ and 10⁶ cells perkg body weight.

In some embodiments, the tolerated error is determined based on thedeviation from the desired average test subjects are monitored to assessthe therapeutic efficacy and/or safety of each percentage combination.In some embodiments, the tolerated error will be within about 1%, about2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50% of thedesired ratio, including any value in between these ranges.

6. Exemplary Methods of Selecting or Enriching Cells

a. Single Process Stream and/or Simultaneous Selection UsingImmunomagnetic Beads

In some embodiments, the separation and/or steps is carried out usingimmunomagnetic beads. In some embodiments, a cell sample containing CD4+and CD8+ cells, such as a primary human T cell sample, is contacted withmagnetic beads containing a first immunoaffinity reagent that binds toCD4 or CD8 and magnetic beads containing a second immunoaffinity reagentthat binds to the other of the CD4 or CD8. The separation and/or stepscan occur simultaneously and/or sequentially.

In some embodiments, contacting the cells with the magnetic beads isperformed simultaneously, whereby enrichment of cells containing thesurface markers CD4 and CD8 also is performed simultaneously. In somesuch aspects, the method includes contacting cells of a samplecontaining primary human T cells with a first immunoaffinity reagentthat specifically binds to CD4 and a second immunoaffinity reagent thatspecifically binds to CD8 in an incubation composition, under conditionswhereby the immunoaffinity reagents specifically bind to CD4 and CD8molecules, respectively, on the surface of cells in the sample, andrecovering cells bound to the first and/or the second immunoaffinityreagent, thereby generating an enriched composition including CD4+ cellsand CD8+ cells at a culture-initiating ratio.

In some embodiments, the first and/or second immunoaffinity reagent arepresent in the incubation composition at a sub-optimal yieldconcentration, whereby the enriched composition contains less than 70%of the total CD4+ cells in the incubation composition and/or less than70% of the CD8+ cells in the incubation composition, thereby producing acomposition enriched for CD4+ and CD8+ T cells.

In some embodiments, the suboptimal yield concentration of the affinityreagent is a concentration below a concentration used or required toachieve an optimal or maximal yield of bound cells in a given selectionor enrichment involving incubating cells with the reagent and recoveringor separating cells having bound to the reagent (“yield,” for example,being the number of the cells so-recovered or selected compared to thetotal number of cells in the incubation that are targeted by the reagentor to which the reagent is specific or that have a marker for which thereagent is specific and capable of binding). The suboptimal yieldconcentration generally is a concentration or amount of the reagent thatin such process or step achieves no more than 70% yield of bound cells,upon recovery of the cells having bound to the reagent. In someembodiments, no more than at or about 50%, 45%, 40%, 30%, or 25% yieldis achieved by the suboptimal concentration. The concentration may beexpressed in terms of number or mass of particles or surfaces per celland/or number of mass or molecules of agent (e.g., antibody, such asantibody fragment) per cell.

For example, in some embodiments, the suboptimal yield concentration isless than at or about 30 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition. In some embodiments, the suboptimal yield concentration isless than at or about 30 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 20 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 10 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 15 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 10 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 5 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 1 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 0.5 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 0.2 μM agent (e.g., antibody) per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition.

In some embodiments, the suboptimal yield concentration is less than ator about 15 mg beads, particles, surface, or total regent, per 1×10⁹,per 2×10⁹ cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per10×10⁹ cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 10 mg beads, particles, surface, or total regent,per 1×10⁹, per 2×10⁹ cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹cells, per 10×10⁹ cells, per 15×10⁹ cells, or per 20×10⁹ cells in theincubation composition; in some embodiments, the suboptimal yieldconcentration is less than at or about 5 mg beads, particles, surface,or total regent, per 1×10⁹, per 2×10⁹ cells, per 3×10⁹ cells, per 4×10⁹cells, per 5×10⁹ cells, per 10×10⁹ cells, per 15×10⁹ cells, or per20×10⁹ cells in the incubation composition; in some embodiments, thesuboptimal yield concentration is less than at or about 4 mg beads,particles, surface, or total regent, per 1×10⁹, per 2×10⁹ cells, per3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹ cells, per15×10⁹ cells, or per 20×10⁹ cells in the incubation composition; in someembodiments, the suboptimal yield concentration is less than at or about3 mg beads, particles, surface, or total regent, per 1×10⁹, per 2×10⁹cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹cells, per 15×10⁹ cells, or per 20×10⁹ cells in the incubationcomposition; in some embodiments, the suboptimal yield concentration isless than at or about 2 mg beads, particles, surface, or total regent,per 1×10⁹, per 2×10⁹ cells, per 3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹cells, per 10×10⁹ cells, per 15×10⁹ cells, or per 20×10⁹ cells in theincubation composition; in some embodiments, the suboptimal yieldconcentration is less than at or about 1 mg beads, particles, surface,or total regent, per 1×10⁹, per 2×10⁹ cells, per 3×10⁹ cells, per 4×10⁹cells, per 5×10⁹ cells, per 10×10⁹ cells, per 15×10⁹ cells, or per20×10⁹ cells in the incubation composition; in some embodiments, thesuboptimal yield concentration is less than at or about 0.5 mg beads,particles, surface, or total regent, per 1×10⁹, per 2×10⁹ cells, per3×10⁹ cells, per 4×10⁹ cells, per 5×10⁹ cells, per 10×10⁹ cells, per15×10⁹ cells, or per 20×10⁹ cells in the incubation composition.

In some embodiments, e.g., when operating in a suboptimal yieldconcentration for each or one or more of two or more selection reagentswith affinity to two or more markers or cells, one or more of suchreagents is used at a concentration that is higher than one or more ofthe other such reagent(s), in order to bias the ratio of the cell typerecognized by that reagent as compared to the cell type(s) recognized bythe other(s). For example, the reagent specifically binding to themarker for which it is desired to bias the ratio may be included at aconcentration (e.g., agent or mass per cells) that is increased by half,1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more, compared toother(s), depending on how much it is desired to increase the ratio.

In some embodiments, employing suboptimal yield concentration to biasone or both populations of cells can achieve a desired or chosenculture-initiation ratio. In some embodiments, the selection isperformed from a sample, which is a sample containing CD4+ and CD8+cells, containing high numbers of cells, such as at least 1×10⁹ cells,2×10⁹ cells, 3×10⁹ cells, 4×10⁹ cells, 5×10⁹ cells, 6×10⁹ cells, 7×10⁹cells, 8×10⁹ cells, 1×10¹⁰ cells, 2×10¹⁰ cells, 3×10¹⁰ cells, 4×10¹⁰cells, 5×10¹⁰ cells or more. In some embodiments, the high number ofcells is sufficient to ensure saturation of the immunoaffinity reagentsin the sample to cells expressing a marker, such as CD4 or CD8, in whichthe reagent specifically binds.

In some embodiments, when operating in the suboptimal range and/or withenough cells to achieve saturation of reagents, the amount ofimmunoaffinity reagent is proportional to the approximate yield ofenriched cells. In one embodiment, to achieve a culture-initiation ratioof about or approximately 1:1 of CD4+ cells to CD8+ cells, selection ofCD4+ cells and CD8+ cells can be performed with the same, or about thesame, suboptimal yield concentration of immunoaffinity reagents to CD4and CD8, respectively. In another exemplary embodiment, to achieve aculture-initiation ratio of about or approximately 2:1 of CD4+ cells toCD8+ cells, selection of CD4+ cells can be performed with a suboptimalyield concentration of an immunoaffinity reagent to CD4 that is about orapproximately two times greater than the suboptimal yield concentrationof an immunoaffinity reagent to CD8. It is within the level of a skilledartisan to empirically select or choose an appropriate amount orconcentration of immunoaffinity reagents depending on the desired orchosen culture-initiating ratio of the generated composition containingenriched or selected cells in view of the above exemplification.

In some embodiments, the separation and/or steps is carried out usingmagnetic beads in which immunoaffinity reagents are reversibly bound,such as via a peptide ligand interaction with a streptavidin mutein asdescribed above. Exemplary of such magnetic beads are Streptamers®. Insome embodiments, the separation and/or steps is carried out usingmagnetic beads, such as those commercially available from MiltenyiBiotec.

In some aspects, the separation and/or other steps is carried out forautomated separation of cells on a clinical-scale level in a closed andsterile system. Components can include an integrated microcomputer,magnetic separation unit, peristaltic pump, and various pinch valves.The integrated computer in some aspects controls all components of theinstrument and directs the system to perform repeated procedures in astandardized sequence. The magnetic separation unit in some aspectsincludes a movable permanent magnet and a holder for the selectioncolumn. The peristaltic pump controls the flow rate throughout thetubing set and, together with the pinch valves, ensures the controlledflow of buffer through the system and continual suspension of cells. Insome aspects, the separation and/or other steps is carried out usingCliniMACS system (Miltenyi Biotic).

In some embodiments, the automated separation, such as using theCliniMACS system, in some aspects uses antibody-coupled magnetizableparticles that are supplied in a sterile, non-pyrogenic solution. Insome embodiments, after labelling of cells with magnetic particles thecells are washed to remove excess particles. A cell preparation bag isthen connected to the tubing set, which in turn is connected to a bagcontaining buffer and a cell collection bag. The tubing set consists ofpre-assembled sterile tubing, including a pre-column and a separationcolumn, and are for single use only. After initiation of the separationprogram, the system automatically applies the cell sample onto theseparation column. Labelled cells are retained within the column, whileunlabeled cells are removed by a series of washing steps. In someembodiments, the cell populations for use with the methods describedherein are unlabeled and are not retained in the column. In someembodiments, the cell populations for use with the methods describedherein are labeled and are retained in the column. In some embodiments,the cell populations for use with the methods described herein areeluted from the column after removal of the magnetic field, and arecollected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried outusing a system equipped with a cell processing unit that permitsautomated washing and fractionation of cells by centrifugation. In someaspects, the separation and/or other steps are carried out using theCliniMACS Prodigy system (Miltenyi Biotec). A system with a cellprocessing unit can also include an onboard camera and image recognitionsoftware that determines the optimal cell fractionation endpoint bydiscerning the macroscopic layers of the source cell product. Forexample, peripheral blood is automatically separated into erythrocytes,white blood cells and plasma layers. A cell processing system, such asthe CliniMACS Prodigy system, can also include an integrated cellcultivation chamber which accomplishes cell culture protocols such as,e.g., cell differentiation and expansion, antigen loading, and long-termcell culture. Input ports can allow for the sterile removal andreplenishment of media and cells can be monitored using an integratedmicroscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) JImmunother. 35(9):689-701.

In some embodiments, a cell population described herein is collected andenriched (or depleted) via flow cytometry, in which cells stained formultiple cell surface markers are carried in a fluidic stream. In someembodiments, a cell population described herein is collected andenriched (or depleted) via preparative scale (FACS)-sorting. In certainembodiments, a cell population described herein is collected andenriched (or depleted) by use of microelectromechanical systems (MEMS)chips in combination with a FACS-based detection system (see, e.g., WO2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al.(2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeledwith multiple markers, allowing for the isolation of well-defined T cellsubsets at high purity.

In some embodiments, the antibodies or binding partners are labeled withone or more detectable marker, to facilitate separation for positiveand/or negative selection. For example, separation may be based onbinding to fluorescently labeled antibodies. In some examples,separation of cells based on binding of antibodies or other bindingpartners specific for one or more cell surface markers are carried in afluidic stream, such as by fluorescence-activated cell sorting (FACS),including preparative scale (FACS) and/or microelectromechanical systems(MEMS) chips, e.g., in combination with a flow-cytometric detectionsystem. Such methods allow for positive and negative selection based onmultiple markers simultaneously.

b. Single Process Stream and/or Sequential Selections UsingImmunoaffinity Chromatography

In some embodiments, the first selection or enrichment of a populationof cells and the second selection and/or enrichment of a population ofcells are performed using immunoaffinity-based reagents that include atleast a first and second affinity chromatography matrix, respectively,having immobilized thereon an antibody. In some embodiments, one or bothof the first and/or second selection can employ a plurality of affinitychromatography matrices and/or antibodies, whereby the plurality ofmatrices and/or antibodies employed for the same selection, i.e. thefirst selection or the second selection, are serially connected. In someembodiments, the affinity chromatography matrix or matrices employed ina first and/or second selection adsorbs or is capable of selecting orenriching at least about 50×10⁶ cells/mL, 100×10⁶ cells/mL, 200×10⁶cells/mL or 400×10⁶ cells/mL. In some embodiments, the adsorptioncapacity can be modulated based on the diameter and/or length of thecolumn. In some embodiments, the culture-initiating ratio of theselected or enriched composition is achieved by choosing a sufficientamount of matrix and/or at a sufficient relative amount to achieve theculture-initiating ratio assuming based on, for example, the adsorptioncapacity of the column or columns for selecting cells.

In one exemplary embodiment, the adsorption capacity of the matrix ormatrices is the same between the first and second selection, e.g. is oris about 1×10⁸ cells/mL for both, whereby enrichment or selection ofcells in the first selection and second selection results in acomposition containing a CD4+ cells to CD8+ cells at aculture-initiating ratio of or about 1:1. In another exemplaryembodiment, the adsorption capacity of the matrix or matrices used inone of the first selection or second selection is at least 1.5-fold,2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold,9.0-fold, 10.0-fold or greater than the adsorption capacity of thematrix or matrices used in the other of the first selection or secondselection, thereby resulting in a culture-initiation ratio in whichcells selected with the greater adsorption capacity, e.g. CD4+ cells orCD8+ cells, are present in the culture initiating ratio in an amountthat is at least 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold,6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold or greater than theother cell population. It is within the level of a skilled artisan toselect or choose an appropriate volume, diameter or number of affinitymatrix chromatography columns for the first and/or second selectiondepending on the desired or chosen culture-initiating ratio of thegenerated composition containing enriched or selected cells.

Exemplary processes for carrying out selections by the provided methodsare set forth in Example 2. In some embodiments, such processes achievea desired culture-initiation ratio in an enriched or generatedcomposition.

In some embodiments, the first and/or second selection in the providedmethods includes first enriching for one of CD4+ or CD8+ cells, and thenenriching for a sub-population of cells based on, for example, surfaceexpression of a marker expressed on resting, nave or central memory Tcells, e.g. a marker that is CD28, CD62L, CCR7, CD127 or CD27. In someembodiments, the first and/or second selection includes enriching forCD8+ cells, said selection further comprises enriching for centralmemory T (T_(CM)) cells, where the other of the first and/or secondselection includes enriching for CD4+ cells. In some embodiment, themethods are performed to enrich or select for CD4+ cells and to enrichor select for a sub-population of cells that are CD8+/CD28+,CD8+/CD62L+, CD8+/CCR7+, CD8+/CD127+ or CD8+/CD27+. In some embodiments,the first selection includes enriching for CD8+ cells and the secondselection includes enriching for CD4+ cells, where the first selection,which includes cells enriched for CD8+ cells, further includes enrichingfor central memory T (T_(CM)) cells or enriching for cells thatexpresses a marker that is CD28, CD62L, CCR7, CD127 or CD27, therebygenerating a composition, such as a culture-initiation composition,containing CD4+ cells and CD8+ enriched for central memory T (T_(CM))cells or a cell expressing a marker that is CD28, CD62L, CCR7, CD127 orCD27. In some embodiments, the first selection includes enriching forCD4+ cells and the second selection includes enriching for CD8+ cells,wherein the second selection, which includes cells enriched for CD8+cells, further includes enriching for central memory T (T_(CM)) cells orenriching for cells that expresses a marker that is CD28, CD62L, CCR7,CD127 or CD27, thereby generating a composition, such as aculture-initiation composition, containing CD4+ cells and CD8+ enrichedfor central memory T (T_(CM)) cells or a cell expressing a marker thatis CD28, CD62L, CCR7, CD127 or CD27.

In some such embodiments involving a further enrichment of asub-population of cells, to achieve the culture-initiating ratio of CD4+cells or a sub-population thereof to CD8+ cells or a sub-populationthereof, the adsorption capacity of a column matrix or matrices isadjusted to account for differences in the frequency of asub-population, i.e. CD4+ or CD8+ cells enriched for resting, nave,central memory cells or cells expressing a marker that is CD28, CD62L,CCR7, CD127 or CD27, compared to the frequency of cells of therespective CD4+ or CD8+ parent population in the starting sample fromthe subject. The relative level or frequency of various cell populationsin a subject can be determined based on assessing surface expression ofa marker or markers present on such populations or sub-populations. Anumber of well-known methods for assessing expression level of surfacemarkers or proteins may be used, such as detection by affinity-basedmethods, e.g., immunoaffinity-based methods, e.g., in the context ofcell surface proteins, such as by flow cytometry.

In an exemplary embodiment, a sample is enriched for CD4+ andCD8+/CD62L+ cells to yield a CD4+ to CD8+ ratio of 1:1. In thisexemplary embodiment, the first selection can include enriching for CD8+cells using a column with an adsorption capacity adjusted for therelative frequencies of CD4+ cells to CD8+/CD62L+ cells known to bepresent in the sample, or using ratios generally estimated to be in suchsamples. For example, the CD62L+ subpopulation of CD8+ cells collectedfrom a human subject can sometimes be about 25% of the total CD8+ T cellfraction. See e.g. Maldonado, Arthritis Res Ther. 2003; 5(2): R91-R96.In such an embodiment, the columns can be arrayed to collect 4-fold moreCD8+ cells than CD4+ cells in order to generate a 1:1 culture initiationratio of the CD4+ cells and the CD8+ sub-population further containingCD62L+ cells. Thus, assuming a similar adsorption capacity andefficiency for each selection column, the CD8+ column can be about orapproximately 4-fold larger than the CD4 selection column or the CD62Lselection column. The size of the columns can also be adjusted forexpected yield. For example, if each column is only 80% efficient, thesize of each column can be adjusted to account for the efficiency ofeach subsequent selection.

For example a desired or chosen culture initiation composition can beone that contains 200×10⁶ CD4+ cells and 200×10⁶ CD8+/CD62L+ cells. Inthis example, assuming the ratios presented above, the sample to beenriched can contain at least or about 200×10⁶ CD4+ cells and at leastor about 800×10⁶ CD8+ cells, approximately 25% of which (200×10⁶) mayalso be CD62L+. Assuming that each 2 mL of selection matrix can enrichfor about or approximately 200×10⁶ cells, a CD8 selection column with 8mL of selection matrix can bind about or approximately 800×10⁶ CD8+cells while the flow-through passes to the CD4 selection column. A CD4selection column with 2 mL of selection matrix can bind about orapproximately 200×10⁶CD4+ cells. The CD8+ cells can be further enrichedfor CD62L by eluting the CD8+ cells into a CD62L selection column. Inthis exemplary embodiment, the CD62L selection column can contain 2 mLof selection matrix, thus enriching for about or approximately 200×10⁶CD8+/CD62L+ cells. The CD4 and CD8/CD62L columns can be eluted into aculture vessel, yielding the initiation culture composition or acomposition having about or approximately a 1:1 culture-initiationratio.

In another exemplary embodiment, a sample is enriched for CD4+ andCD8+/CCR7+ cells to yield a CD4+ to CD8+ ratio of 1:1. In this exemplaryembodiment, the first selection can include enriching for CD8+ cellsusing a column with an adsorption capacity adjusted for the relativefrequencies of CD4+ cells to CD8+/CCR7+ cells known to be present in thesample, or using ratios generally estimated to be in such samples. Forexample, the CCR7+ subpopulation of CD8+ cells collected from a humansubject can sometimes be about 60% of the total CD8+ T cell fraction.See e.g. Chen, Blood. 2001 Jul. 1; 98(1):156-64. The columns can bearrayed to collect 3⅓-fold more CD8+ cells than CD4+ cells in order togenerate a 1:1 culture initiation ratio. Assuming a similar adsorptioncapacity and efficiency for each selection column, the CD8+ column canbe about or approximately 3⅓-fold larger than the CD4 selection columnor the CCR7 selection column. The size of the columns can also beadjusted for expected yield. For example, if each column is only 80%efficient, the size of each column can be adjusted to account for theefficiency of each subsequent selection.

For example, a desired initiation culture can contain 200×10⁶ CD4+ cellsand 200×10⁶ CD8+/CCR7+ cells. In this example, assuming the ratiospresented above, the sample to be enriched can contain at least 200×10⁶CD4+ cells and at least or about 6.6×10⁶ CD8+ cells, approximately 60%of which (200×10⁶) may also be CCR7+. Assuming that each 2 mL ofselection matrix can enrich for 200×10⁶ cells, a CD8 selection columnwith 3⅓ mL of selection matrix can bind about or approximately 6.6×10⁶CD8+ cells while the flow-through passes to the CD4 selection column. ACD4 selection column with 2 mL of selection matrix can bind about orapproximately 200×10⁶CD4+ cells. The CD8+ cells can be further enrichedfor CD62L by eluting the CD8+ cells into a CCR7 selection column. Inthis exemplary embodiment, the CCR7 selection column can contain 1 mL ofselection matrix, thus enriching for about or approximately 200×10⁶CD8+/CCR7+ cells. The CD4 and CCCR7 columns can be eluted into a culturevessel, yielding the initiation culture containing about orapproximately 200×10⁶ CD4+ cells and about or approximately 200×10⁶CD8+/CCR7+ cells, or a 1:1 culture-initiation ratio.

It is within the level of a skilled artisan to empirically select orchoose an appropriate volume, diameter or number of affinity matrixchromatography columns for the first and/or second and/or thirdselection depending on the desired or chosen culture-initiating ratio ofthe generated composition containing enriched or selected cells, theexpected frequency of each sub-population, the varying efficiencies ofeach selection column and other factors within the level of the skilledartisan and in view of the above exemplification.

B. Incubation of Isolated Cells

In some embodiments, the provided methods include one or more of varioussteps for incubating isolated cells and cell populations, such aspopulations isolated according to the methods herein, such as steps forincubating an isolated CD4+ T cell population, e.g., unfractionated CD4+T cell population or subpopulation(s) thereof, and an isolated CD8+ Tcell population, e.g., isolated unfractionated CD8+ T cell population orsubpopulation(s) thereof. In some embodiments, the cell populations areincubated in a culture-initiating composition.

The plurality of isolated cell populations, e.g., the CD4+ and CD8+ cellpopulations (e.g., unfractionated or subpopulations thereof) aregenerally incubated with the cell populations combined in aculture-initiating composition in the same culture vessel, such as thesame unit, chamber, well, column, tube, tubing set, valve, vial, culturedish, bag, or other container for culture or cultivating cells.

In some aspects, the cell populations or cell types are present in theculture-initiating composition at a culture-initiating ratio, e.g.,ratio of CD4⁺ and CD8⁺ cells, designed to achieve a particular desiredoutput ratio, or a ratio that is within a certain range of toleratederror of such a desired output ratio, following the incubation and/orengineering steps, or designed to do so a certain percentage of thetime. The output ratio, for example, can be a ratio optimal forachieving one or more therapeutic effects upon administration to apatient, e.g., via adoptive cell therapy. In some aspects, theculture-initiating ratio is determined empirically, e.g., using adetermination method as described herein, for example, to determine theoptimal culture-initiating ratio for achieving a desired output ratio ina particular context.

The incubation steps can include culture, cultivation, stimulation,activation, propagation, including by incubation in the presence ofstimulating conditions, for example, conditions designed to induceproliferation, expansion, activation, and/or survival of cells in thepopulation, to mimic antigen exposure, and/or to prime the cells forgenetic engineering, such as for the introduction of a geneticallyengineered antigen receptor.

The conditions can include one or more of particular media, temperature,oxygen content, carbon dioxide content, time, agents, e.g., nutrients,amino acids, antibiotics, ions, and/or stimulatory factors, such ascytokines, chemokines, antigens, binding partners, fusion proteins,recombinant soluble receptors, and any other agents designed to activatethe cells. In one example, the stimulating conditions include one ormore agent, e.g., ligand, which turns on or initiates TCR/CD3intracellular signaling cascade in a T cell. Such agents can includeantibodies, such as those specific for a TCR component and/orcostimulatory receptor, e.g., anti-CD3, anti-CD28, anti-4-1BB, forexample, bound to solid support such as a bead, and/or one or morecytokines. Optionally, the expansion method may further comprise thestep of adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least about 0.5 ng/ml). Optionally, theexpansion method may further comprise the step of adding IL-2 and/orIL-15 and/or IL-7 and/or IL-21 to the culture medium (e.g., wherein theconcentration of IL-2 is at least about 10 units/m 1).

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177 to Riddell et al.,Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al.(2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, the cell populations, such as CD4⁺ and CD8⁺populations or subpopulations, are expanded by adding to theculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells contains at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). In some aspects, thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads to prevent cell division. In someaspects, the feeder cells are added to culture medium prior to theaddition of the populations of T cells.

In some embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at or about 37 degrees Celsius. In some embodiments, atemperature shift is effected during culture, such as from 37 degreesCelsius to 35 degrees Celsius. Optionally, the incubation may furthercomprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL)as feeder cells. LCL can be irradiated with gamma rays in the range ofabout 6000 to 10,000 rads. The LCL feeder cells in some aspects isprovided in any suitable amount, such as a ratio of LCL feeder cells toinitial T lymphocytes of at least about 10:1.

In embodiments, populations of CD4⁺ and CD8⁺ that are antigen specificcan be obtained by stimulating naive or antigen specific T lymphocyteswith antigen. For example, antigen-specific T cell lines or clones canbe generated to cytomegalovirus antigens by isolating T cells frominfected subjects and stimulating the cells in vitro with the sameantigen. Naive T cells may also be used.

Interim Assessment and Adjustment

In some embodiments, the methods include assessment and/or adjustment ofthe cells or composition containing the cells, at a time subsequent tothe initiation of the incubation or culture, such as at a time duringthe incubation. Assessment can include taking one or more measurementsof a composition or vessel containing the cells, such as assessing cellsfor proliferation rate, degree of survival, phenotype, e.g., expressionof one or more surface or intracellular markers, such as proteins orpolynucleotides, and/or assessing the composition or vessel fortemperature, media component(s), oxygen or carbon dioxide content,and/or presence or absence or amount or relative amount of one or morefactors, agents, components, and/or cell types, including subtypes.Assessment in some embodiments includes determining an intermediateratio of a plurality, e.g., two cell types, such as CD4⁺ and CD8⁺ Tcells, in the composition or vessel being incubated. In some aspects,the assessment is performed in an automated fashion, for example, usinga device as described herein, and/or is set ahead of time to be carriedout at certain time-points during incubation. In some aspects, theoutcome of the assessment, such as a determined interim ratio of twotypes of cells, indicates that an adjustment should be made, such asaddition or removal of one or more cell types.

Adjustment can include adjusting any cell culture factor or parameter,such as temperature, length (time) for which incubation or a stepthereof will be carried out (duration of incubation), replenishment,addition and/or removal of one or more components in the compositionbeing incubated, e.g., media or buffer or components thereof, agents,e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatoryfactors, such as cytokines, chemokines, antigens, binding partners,fusion proteins, recombinant soluble receptors, or cells or cell typesor populations of cells. In some aspects, the removal or addition ofvarious components or other adjustment is carried out in an automatedfashion, for example, using a device or system as described herein. Insome embodiments, the system is programmed such that an adjustment isautomatically initiated based on a certain readout from an interimassessment. For example, in some cases, a system or device is programmedto carry out one or more assessments at a particular time; the system ordevice in such cases can be further programmed such that a particularoutcome of such an assessment, such as a particular ratio of one celltype to another, initiates a particular adjustment, such as addition ofone or more of the cell types.

In some aspects, the adjustment is carried out by addition or removal ina way that does not disrupt a closed environment containing the cellsand compositions, such as by input and/or removal valves, designed toadd or remove components while maintaining sterility, such as in one ormore device or system as described herein.

In a particular embodiment, an interim ratio of CD4⁺ to CD8⁺ T cells isassessed during the incubation period. In some embodiments, theassessment is carried out after 1, 2, 3, 4, 5, 6, or 7 days, such asbetween 3 and 5 days, and/or at a time point in which all cells are inor suspected of being in cell cycle. In some aspects, the interim ratioso-determined indicates that CD4⁺ or CD8⁺ T cells, such as cells of theisolated population of CD4⁺ T cells or isolated population of CD8⁺ Tcells (e.g., sub-population, such as central memory CD8⁺ T cells),should be added to or enriched in the culture vessel or to thecomposition being incubated. Thus, in some aspects, the assessment isfollowed by such an addition or removal, typically an addition. In someaspects, multiple assessments and possible adjustments are carried outover the course of incubation, e.g., in an iterative fashion.

In some embodiments, where cells are engineered, e.g., to introduce agenetically engineered antigen receptor, the incubation in the presenceof one or more stimulating agents continues during the engineeringphase.

In some embodiments, the cells are incubated for at or about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, either in total or prior toengineering.

C. Engineering, Engineered Antigen Receptors, and Engineered Cells

In some embodiments, the methods include genetic engineering of theisolated and/or incubated cells, such as to introduce into the cellsrecombinant genes for expression of molecules, such as receptors, e.g.,antigen receptors, useful in the context of adoptive therapy.

Among the genes for introduction are those to improve the efficacy oftherapy, such as by promoting viability and/or function of transferredcells; genes to provide a genetic marker for selection and/or evaluationof the cells, such as to assess in vivo survival or localization; genesto improve safety, for example, by making the cell susceptible tonegative selection in vivo as described by Lupton S. D. et al., Mol. andCell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy3:319-338 (1992); see also the publications of PCT/US91/08442 andPCT/US94/05601 by Lupton et al. describing the use of bifunctionalselectable fusion genes derived from fusing a dominant positiveselectable marker with a negative selectable marker. This can be carriedout in accordance with known techniques (see, e.g., Riddell et al., U.S.Pat. No. 6,040,177, at columns 14-17) or variations thereof that will beapparent to those skilled in the art based upon the present disclosure.

The engineering generally includes introduction of gene or genes forexpression of a genetically engineered antigen receptor. Among suchantigen receptors are genetically engineered T cell receptors (TCRs) andcomponents thereof, and functional non-TCR antigen receptors, such aschimeric antigen receptors (CAR).

The antigen receptor in some embodiments specifically binds to a ligandon a cell or disease to be targeted, such as a cancer or other diseaseor condition, including those described herein for targeting with theprovided methods and compositions. Exemplary antigens are orphantyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22,mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor,CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2,3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA,IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-celladhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2DLigands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72,VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen,PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2,CD123, CS-1, c-Met, GD-2, and MAGE A3 and/or biotinylated molecules,and/or molecules expressed by HIV, HCV, HBV or other pathogens.

Antigen Receptors

In one embodiment, the engineered antigen receptors are CARs. The CARsgenerally include genetically engineered receptors including anextracellular ligand binding domain linked to one or more intracellularsignaling components. Such molecules typically mimic or approximate asignal through a natural antigen receptor and/or signal through such areceptor in combination with a costimulatory receptor.

In some embodiments, CARs are constructed with specificity for aparticular marker, such as a marker expressed in a particular cell typeto be targeted by adoptive therapy, e.g., a cancer marker. This isachieved in some aspects by inclusion in the extracellular portion ofthe CAR one or more antigen binding molecule, such as one or moreantigen-binding fragment, domain, or portion, or one or more antibodyvariable domains, and/or antibody molecules. In some embodiments, theCAR includes an antigen-binding portion or portions of an antibodymolecule, such as a single-chain antibody fragment (scFv) derived fromthe variable heavy (VH) and variable light (VL) chains of a monoclonalantibody (mAb).

In some embodiments, the CAR comprises an antibody heavy chain domainthat specifically binds a cell surface antigen of a cell or disease tobe targeted, such as a tumor cell or a cancer cell, such as any of thetarget antigens described herein or known in the art.

In some embodiments, the tumor antigen or cell surface molecule is apolypeptide. In some embodiments, the tumor antigen or cell surfacemolecule is selectively expressed or overexpressed on tumor cells ascompared to non-tumor cells of the same tissue.

In some embodiments, the CAR binds a pathogen-specific antigen. In someembodiments, the CAR is are specific for viral antigens (such as HIV,HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

In some aspects, the antigen-specific binding, or recognition componentis linked to one or more transmembrane and intracellular signalingdomains. In some embodiments, the CAR includes a transmembrane domainfused to the extracellular domain of the CAR. In one embodiment, thetransmembrane domain that naturally is associated with one of thedomains in the CAR is used. In some instances, the transmembrane domainis selected or modified by amino acid substitution to avoid binding ofsuch domains to the transmembrane domains of the same or differentsurface membrane proteins to minimize interactions with other members ofthe receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain in some aspects is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154. Alternatively the transmembrane domain in some embodiments issynthetic. In some aspects, the synthetic transmembrane domain comprisespredominantly hydrophobic residues such as leucine and valine. In someaspects, a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain.

In some embodiments, a short oligo- or polypeptide linker, for example,a linker of between 2 and 10 amino acids in length, such as onecontaining glycines and serines, e.g., glycine-serine doublet, ispresent and forms a linkage between the transmembrane domain and thecytoplasmic signaling domain of the CAR.

The CAR generally includes intracellular signaling component orcomponents. In some embodiments, the CAR includes an intracellularcomponent of the TCR complex, such as a TCR CD3⁺ chain that mediatesT-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in someaspects, the antigen binding molecule is linked to one or more cellsignaling modules. In some embodiments, cell signaling modules includeCD3 transmembrane domain, CD3 intracellular signaling domains, and/orother CD transmembrane domains. In some embodiments, the CAR furtherincludes a portion of one or more additional molecules such as Fcreceptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, theCAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptorγ and CD8, CD4, CD25 or CD16.

In some embodiments, upon ligation of the CAR, the cytoplasmic domain orintracellular signaling domain of the CAR activates at least one of thenormal effector functions of the immune cell, e.g., T cell engineered toexpress the cell. For example, in some contexts, the CAR induces afunction of a T cell such as cytolytic activity or T-helper activity,such as secretion of cytokines or other factors. In some embodiments, atruncated portion of an intracellular signaling domain of an antigenreceptor component or costimulatory molecule. Such truncated portion insome aspects is used in place of an intact immunostimulatory chain, forexample, if it transduces the effector function signal. In someembodiments, the intracellular signaling domain or domains include thecytoplasmic sequences of the T cell receptor (TCR), and in some aspectsalso those of co-receptors that in the natural context act in concertwith such receptor to initiate signal transduction following antigenreceptor engagement, and/or any derivative or variant of such molecules,and/or any synthetic sequence that has the same functional capability.

In the context of a natural TCR, full activation generally requires notonly signaling through the TCR, but also a costimulatory signal. Thus,in some embodiments, to promote full activation, a component forgenerating secondary or co-stimulatory signal is also included in theCAR. T cell activation is in some aspects described as being mediated bytwo classes of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In some aspects, theCAR includes one or both of such signaling components.

Primary cytoplasmic signaling sequences can in some aspects regulateprimary activation of the TCR complex either in a stimulatory way, or inan inhibitory way. Primary cytoplasmic signaling sequences that act in astimulatory manner may contain signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs or ITAMs. Examples ofITAM containing primary cytoplasmic signaling sequences include thosederived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments,cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmicsignaling domain, portion thereof, or sequence derived from CD3 zeta.

In some embodiments, the CAR includes a signaling domain and/ortransmembrane portion of a costimulatory receptor, such as CD28, 4-1BB,OX40, DAP10, and ICOS.

In certain embodiments, the intracellular signaling domain comprises aCD28 transmembrane and signaling domain linked to a CD3 intracellulardomain. In some embodiments, the intracellular signaling domaincomprises a chimeric CD28 and CD137 co-stimulatory domains, linked to aCD3 intracellular domain. In some embodiments, a CAR can also include atransduction marker (e.g., tEGFR). In some embodiments, theintracellular signaling domain of the CD8⁺ cytotoxic T cells is the sameas the intracellular signaling domain of the CD4⁺ helper T cells. Insome embodiments, the intracellular signaling domain of the CD8⁺cytotoxic T cells is different than the intracellular signaling domainof the CD4⁺ helper T cells.

In some embodiments, the CAR encompasses two or more costimulatorydomain combined with an activation domain, e.g., primary activationdomain, in the cytoplasmic portion. One example is a receptor includingintracellular components of CD3-zeta, CD28, and 4-1BB.

CARs and production and introduction thereof can include thosedescribed, for example, by published patent disclosures WO200014257,U.S. Pat. No. 6,451,995, US2002131960, U.S. Pat. Nos. 7,446,190,8,252,592, EP2537416, US2013287748, and WO2013126726, and/or thosedescribed by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398;Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin.Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March18(2): 160-75.

In some embodiments, the T cells are modified with a recombinant T cellreceptor. In some embodiments, the recombinant TCR is specific for anantigen, generally an antigen present on a target cell, such as atumor-specific antigen, an antigen expressed on a particular cell typeassociated with an autoimmune or inflammatory disease, or an antigenderived from a viral pathogen or a bacterial pathogen.

In some embodiments, the T cells are engineered to express T-cellreceptors (TCRs) cloned from naturally occurring T cells. In someembodiments, a high-affinity T cell clone for a target antigen (e.g., acancer antigen) is identified, isolated from a patient, and introducedinto the cells. In some embodiments, the TCR clone for a target antigenhas been generated in transgenic mice engineered with human immunesystem genes (e.g., the human leukocyte antigen system, or HLA). See,e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin CancerRes. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808. Insome embodiments, phage display is used to isolate TCRs against a targetantigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395and Li (2005) Nat Biotechnol. 23:349-354.

In some embodiments, after the T-cell clone is obtained, the TCR alphaand beta chains are isolated and cloned into a gene expression vector.In some embodiments, the TCR alpha and beta genes are linked via apicornavirus 2A ribosomal skip peptide so that both chains arecoexpression. In some embodiments, genetic transfer of the TCR isaccomplished via retroviral or lentiviral vectors, or via transposons(see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of theAmerican Society of Gene Therapy. 13:1050-1063; Frecha et al. (2010)Molecular Therapy: The Journal of the American Society of Gene Therapy.18:1748-1757; an Hackett et al. (2010) Molecular Therapy: The Journal ofthe American Society of Gene Therapy. 18:674-683.

In some embodiments, gene transfer is accomplished by first stimulatingT cell growth and the activated cells are then transduced and expandedin culture to numbers sufficient for clinical applications.

In some contexts, overexpression of a stimulatory factor (for example, alymphokine or a cytokine) may be toxic to a subject. Thus, in somecontexts, the engineered cells include gene segments that cause thecells to be susceptible to negative selection in vivo, such as uponadministration in adoptive immunotherapy. For example in some aspects,the cells are engineered so that they can be eliminated as a result of achange in the in vivo condition of the patient to which they areadministered. The negative selectable phenotype may result from theinsertion of a gene that confers sensitivity to an administered agent,for example, a compound. Negative selectable genes include the Herpessimplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al.,Cell II: 223, I977) which confers ganciclovir sensitivity; the cellularhypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase,(Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some aspects, the cells further are engineered to promote expressionof cytokines, such as proinflammatory cytokines, e.g., IL-2, IL-12,IL-7, IL-15, IL-21.

Introduction of the Genetically Engineered Components

Various methods for the introduction of genetically engineeredcomponents, e.g., antigen receptors, e.g., CARs, are well known and maybe used with the provided methods and compositions. Exemplary methodsinclude those for transfer of nucleic acids encoding the receptors,including via viral, e.g., retroviral or lentiviral, transduction,transposons, and electroporation.

In some embodiments, recombinant nucleic acids are transferred intocells using recombinant infectious virus particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In some embodiments, recombinant nucleicacids are transferred into T cells using recombinant lentiviral vectorsor retroviral vectors, such as gamma-retroviral vectors (see, e.g.,Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25;Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al.(2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011November; 29(11): 550-557.

In some embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In someembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3:102-109.

Methods of lentiviral transduction are known. Exemplary methods aredescribed in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701;Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009)Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood.102(2): 497-505.

In some embodiments, recombinant nucleic acids are transferred into Tcells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids aretransferred into T cells via transposition (see, e.g., Manuri et al.(2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec TherNucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic materialin immune cells include calcium phosphate transfection (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.), protoplast fusion, cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNAco-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

In some embodiments, the same CAR is introduced into each of CD4⁺ andCD8⁺ T lymphocytes. In some embodiments, a different CAR is introducedinto each of CD4⁺ and CD8⁺ T lymphocytes. In some embodiments, the CARin each of these populations has an antigen binding molecule thatspecifically binds to the same antigen. In some embodiments, the CAR ineach of these populations binds to a different molecule. In someembodiments, the CAR in each of these populations has cellular signalingmodules that differ. In some embodiments each of the CD4⁺ or CD8⁺ Tlymphocytes have been sorted in to naive, central memory, effectormemory or effector cells prior to transduction.

In other embodiments, the cells, e.g., T cells, are not engineered toexpress recombinant receptors, but rather include naturally occurringantigen receptors specific for desired antigens, such astumor-infiltrating lymphocytes and/or T cells cultured in vitro or exvivo, e.g., during the incubation step(s), to promote expansion of cellshaving particular antigen specificity. For example, in some embodiments,the cells are produced for adoptive cell therapy by isolation oftumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes(TIL). The direct targeting of human tumors using autologous tumorinfiltrating lymphocytes can in some cases mediate tumor regression (seeRosenberg S A, et al. (1988) N Engl J Med. 319:1676-1680). In someembodiments, lymphocytes are extracted from resected tumors. In someembodiments, such lymphocytes are expanded in vitro. In someembodiments, such lymphocytes are cultured with lymphokines (e.g.,IL-2). In some embodiments, such lymphocytes mediate specific lysis ofautologous tumor cells but not allogeneic tumor or autologous normalcells.

In some aspects, the incubation and/or engineering steps and/or themethods generally result in a desired output ratio (or a ratio that iswithin a tolerated error or difference from such a ratio), or do sowithin a certain percentage of the time that the methods are performed.

D. Cryopreservation

In some embodiments, the provided methods include steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. In some embodiments, the freeze andsubsequent thaw step removes granulocytes and, to some extent, monocytesin the cell population. In some embodiments, the cells are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This is then diluted 1:1 with media so that the finalconcentration of DMSO and HSA are 10% and 4%, respectively. The cellsare then frozen to 80° C. at a rate of 1° per minute and stored in thevapor phase of a liquid nitrogen storage tank.

II. KITS AND SYSTEMS

Also provided are systems, apparatuses, and kits useful in performingthe provided methods. In one example, a single system is provided whichcarries out one or more of the isolation, cell preparation, separation,e.g., separation based on density, affinity, sensitivity to one or morecomponents, or other property, washing, processing, incubation, culture,and/or formulation steps of the methods. In some aspects, the system isused to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inInternational Patent Application, Publication Number WO2009/072003, orUS 20110003380 A1.

In some embodiments, the system or apparatus carries out one or more,e.g., all, of the isolation, processing, engineering, and formulationsteps in an integrated or self-contained system, and/or in an automatedor programmable fashion. In some aspects, the system or apparatusincludes a computer and/or computer program in communication with thesystem or apparatus, which allows a user to program, control, assess theoutcome of, and/or adjust various aspects of the processing, isolation,engineering, and formulation steps.

Also provided are kits for carrying out the provided methods. In someembodiments, the kits include antibodies or other binding partners,generally coupled to solid supports, for the isolation, e.g., forimmunoaffinity-based separation steps, of the methods.

In some embodiments, the kit comprises antibodies for positive andnegative selection, bound to magnetic beads. In one embodiment, the kitcomprises instructions to carry out selection starting with a sample,such as a PBMC sample, by selecting based on expression of a firstsurface marker, recognized by one or more of the antibodies providedwith the kit, retaining both positive and negative fractions. In someaspects, the instructions further include instructions to carry out oneor more additional selection steps, starting with the positive and/ornegative fractions derived therefrom, for example, while maintaining thecompositions in a contained environment and/or in the same separationvessel.

In one embodiment, a kit comprises anti-CD4, anti-CD14, anti-CD45RA,anti-CD14, and anti-CD62L antibodies, bound to magnetic beads. In oneembodiment, the kit comprises instructions to carry out selectionstarting with a sample, such as a PBMC sample, by selecting based on CD4expression, retaining both positive and negative fractions, and on thenegative fraction, further subjecting the fraction to a negativeselection using the anti-CD14, anti-CD45RA antibodies, and a positiveselection using the anti-CD62L antibody, in either order. Alternatively,the components and instructions are adjusted according to any of theseparation embodiments described herein.

In some embodiments, the kit further includes instructions to transferthe cells of the populations isolated by the selection steps to aculture, cultivation, or processing vessel, while maintaining the cellsin a self-contained system. In some embodiments, the kit includesinstructions to transfer the different isolated cells at a particularratio.

III. CELLS, COMPOSITIONS, AND METHODS OF ADMINISTRATION

Also provided are cells, cell populations, and compositions (includingpharmaceutical and therapeutic compositions) containing the cells andpopulations, produced by the provided methods. Also provided aremethods, e.g., therapeutic methods for administrating the cells andcompositions to subjects, e.g., patients.

Provided are methods of administering the cells, populations, andcompositions, and uses of such cells, populations, and compositions totreat or prevent diseases, conditions, and disorders, including cancers.In some embodiments, the cells, populations, and compositions areadministered to a subject or patient having the particular disease orcondition to be treated, e.g., via adoptive cell therapy, such asadoptive T cell therapy. In some embodiments, cells and compositionsprepared by the provided methods, such as engineered compositions andend-of-production compositions following incubation and/or otherprocessing steps, are administered to a subject, such as a subjecthaving or at risk for the disease or condition. In some aspects, themethods thereby treat, e.g., ameliorate one or more symptom of, thedisease or condition, such as by lessening tumor burden in a cancerexpressing an antigen recognized by an engineered T cell.

Methods for administration of cells for adoptive cell therapy are knownand may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by autologous transfer, in which the cells are isolatedand/or otherwise prepared from the subject who is to receive the celltherapy, or from a sample derived from such a subject. Thus, in someaspects, the cells are derived from a subject, e.g., patient, in need ofa treatment and the cells, following isolation and processing areadministered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, in which the cells are isolatedand/or otherwise prepared from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the cells then are administered to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject.

In some embodiments, the subject, e.g., patient, to whom the cells, cellpopulations, or compositions are administered is a mammal, typically aprimate, such as a human. In some embodiments, the primate is a monkeyor an ape. The subject can be male or female and can be any suitableage, including infant, juvenile, adolescent, adult, and geriatricsubjects. In some embodiments, the subject is a non-primate mammal, suchas a rodent.

Also provided are pharmaceutical compositions for use in such methods.

Among the diseases, conditions, and disorders for treatment with theprovided compositions, cells, methods and uses are tumors, includingsolid tumors, hematologic malignancies, and melanomas, and infectiousdiseases, such as infection with a virus or other pathogen, e.g., HIV,HCV, HBV, CMV, and parasitic disease. In some embodiments, the diseaseor condition is a tumor, cancer, malignancy, neoplasm, or otherproliferative disease. Such diseases include but are not limited toleukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL,non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma,refractory follicular lymphoma, mantle cell lymphoma, indolent B celllymphoma, B cell malignancies, cancers of the colon, lung, liver,breast, prostate, ovarian, skin (including melanoma), bone, and braincancer, ovarian cancer, epithelial cancers, renal cell carcinoma,pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma,colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma,medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.

In some embodiments, the disease or condition is an infectious diseaseor condition, such as, but not limited to, viral, retroviral, bacterial,and protozoal infections, immunodeficiency, Cytomegalovirus (CMV),Epstein-Ban virus (EBV), adenovirus, BK polyomavirus. In someembodiments, the disease or condition is an autoimmune or inflammatorydisease or condition, such as arthritis, e.g., rheumatoid arthritis(RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatorybowel disease, psoriasis, scleroderma, autoimmune thyroid disease,Grave's disease, Crohn's disease multiple sclerosis, asthma, and/or adisease or condition associated with transplant.

In some embodiments, the antigen associated with the disease or disorderis selected from the group consisting of orphan tyrosine kinase receptorROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, andhepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30,CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetalacethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha,IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesionmolecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands,NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,carcinoembryonic antigen (CEA), prostate specific antigen, PSMA,Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,CS-1, c-Met, GD-2, and MAGE A3 and/or biotinylated molecules, and/ormolecules expressed by HIV, HCV, HBV or other pathogens.

In some embodiments, the cells and compositions are administered to asubject in the form of a pharmaceutical composition, such as acomposition comprising the cells or cell populations and apharmaceutically acceptable carrier or excipient. The pharmaceuticalcompositions in some embodiments additionally comprise otherpharmaceutically active agents or drugs, such as chemotherapeuticagents, e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. Insome embodiments, the agents are administered in the form of a salt,e.g., a pharmaceutically acceptable salt. Suitable pharmaceuticallyacceptable acid addition salts include those derived from mineral acids,such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric,and sulphuric acids, and organic acids, such as tartaric, acetic,citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic,and arylsulphonic acids, for example, p-toluenesulphonic acid.

The choice of carrier will in the pharmaceutical composition isdetermined in part by the particular engineered CAR or TCR, vector, orcells expressing the CAR or TCR, as well as by the particular methodused to administer the vector or host cells expressing the CAR.Accordingly, there are a variety of suitable formulations. For example,the pharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In some aspects, a mixtureof two or more preservatives is used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition.

In addition, buffering agents in some aspects are included in thecomposition. Suitable buffering agents include, for example, citricacid, sodium citrate, phosphoric acid, potassium phosphate, and variousother acids and salts. In some aspects, a mixture of two or morebuffering agents is used. The buffering agent or mixtures thereof aretypically present in an amount of about 0.001% to about 4% by weight ofthe total composition. Methods for preparing administrablepharmaceutical compositions are known. Exemplary methods are describedin more detail in, for example, Remington: The Science and Practice ofPharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

In certain embodiments, the pharmaceutical composition is formulated asan inclusion complex, such as cyclodextrin inclusion complex, or as aliposome. Liposomes can serve to target the host cells (e.g., T-cells orNK cells) to a particular tissue. Many methods are available forpreparing liposomes, such as those described in, for example, Szoka etal., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos.4,235,871, 4,501,728, 4,837,028, and 5,019,369.

In some embodiments, the pharmaceutical composition employstime-released, delayed release, and/or sustained release deliverysystems, such that the delivery of the composition occurs prior to, andwith sufficient time to cause, sensitization of the site to be treated.Many types of release delivery systems are available and known to thoseof ordinary skill in the art. Such systems in some aspects can avoidrepeated administrations of the composition, thereby increasingconvenience to the subject and the physician.

In some embodiments, the he pharmaceutical composition comprises thecells or cell populations in an amount that is effective to treat orprevent the disease or condition, such as a therapeutically effective orprophylactically effective amount. Thus, in some embodiments, themethods of administration include administration of the cells andpopulations at effective amounts. Therapeutic or prophylactic efficacyin some embodiments is monitored by periodic assessment of treatedsubjects. For repeated administrations over several days or longer,depending on the condition, the treatment is repeated until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful and can be determined. The desired dosage can be deliveredby a single bolus administration of the composition, by multiple bolusadministrations of the composition, or by continuous infusionadministration of the composition.

In some embodiments, the cells are administered at a desired dosage,which in some aspects includes a desired dose or number of cells or celltype(s) and/or a desired ratio of cell types. Thus, the dosage of cellsin some embodiments is based on a total number of cells (or number perkg body weight) and a desired ratio of the individual populations orsub-types, such as the CD4+ to CD8+ ratio. In some embodiments, thedosage of cells is based on a desired total number (or number per kg ofbody weight) of cells in the individual populations or of individualcell types. In some embodiments, the dosage is based on a combination ofsuch features, such as a desired number of total cells, desired ratio,and desired total number of cells in the individual populations.

In some embodiments, the populations or sub-types of cells, such as CD8⁺and CD4⁺ T cells, are administered at or within a tolerated differenceof a desired dose of total cells, such as a desired dose of T cells. Insome aspects, the desired dose is a desired number of cells or a desirednumber of cells per unit of body weight of the subject to whom the cellsare administered, e.g., cells/kg. In some aspects, the desired dose isat or above a minimum number of cells or minimum number of cells perunit of body weight. In some aspects, among the total cells,administered at the desired dose, the individual populations orsub-types are present at or near a desired output ratio (such as CD4⁺ toCD8⁺ ratio), e.g., within a certain tolerated difference or error ofsuch a ratio.

In some embodiments, the cells are administered at or within a tolerateddifference of a desired dose of one or more of the individualpopulations or sub-types of cells, such as a desired dose of CD4+ cellsand/or a desired dose of CD8+ cells. In some aspects, the desired doseis a desired number of cells of the sub-type or population, or a desirednumber of such cells per unit of body weight of the subject to whom thecells are administered, e.g., cells/kg. In some aspects, the desireddose is at or above a minimum number of cells of the population orsub-type, or minimum number of cells of the population or sub-type perunit of body weight.

Thus, in some embodiments, the dosage is based on a desired fixed doseof total cells and a desired ratio, and/or based on a desired fixed doseof one or more, e.g., each, of the individual sub-types orsub-populations. Thus, in some embodiments, the dosage is based on adesired fixed or minimum dose of T cells and a desired ratio of CD4⁺ toCD8⁺ cells, and/or is based on a desired fixed or minimum dose of CD4⁺and/or CD8⁺ cells.

In certain embodiments, the cells, or individual populations ofsub-types of cells, are administered to the subject at a range of aboutone million to about 100 billion cells, such as, e.g., 1 million toabout 50 billion cells (e.g., about 5 million cells, about 25 millioncells, about 500 million cells, about 1 billion cells, about 5 billioncells, about 20 billion cells, about 30 billion cells, about 40 billioncells, or a range defined by any two of the foregoing values), such asabout 10 million to about 100 billion cells (e.g., about 20 millioncells, about 30 million cells, about 40 million cells, about 60 millioncells, about 70 million cells, about 80 million cells, about 90 millioncells, about 10 billion cells, about 25 billion cells, about 50 billioncells, about 75 billion cells, about 90 billion cells, or a rangedefined by any two of the foregoing values), and in some cases about 100million cells to about 50 billion cells (e.g., about 120 million cells,about 250 million cells, about 350 million cells, about 450 millioncells, about 650 million cells, about 800 million cells, about 900million cells, about 3 billion cells, about 30 billion cells, about 45billion cells) or any value in between these ranges.

In some embodiments, the dose of total cells and/or dose of individualsub-populations of cells is within a range of between at or about 10⁴and at or about 10⁹ cells/kilograms (kg) body weight, such as between10⁵ and 10⁶ cells/kg body weight, for example, at or about 1×10⁵cells/kg, 1.5×10⁵ cells/kg, 2×10⁵ cells/kg, or 1×10⁶ cells/kg bodyweight. For example, in some embodiments, the cells are administered at,or within a certain range of error of, between at or about 10⁴ and at orabout 10⁹ T cells/kilograms (kg) body weight, such as between 10⁵ and10⁶ T cells/kg body weight, for example, at or about 1×10⁵ T cells/kg,1.5×10⁵ T cells/kg, 2×10⁵ T cells/kg, or 1×10⁶ T cells/kg body weight.

In some embodiments, the cells are administered at or within a certainrange of error of between at or about 10⁴ and at or about 10⁹ CD4⁺and/or CD8⁺ cells/kilograms (kg) body weight, such as between 10⁵ and10⁶ CD4⁺ and/or CD8⁺ cells/kg body weight, for example, at or about1×10⁵ CD4⁺ and/or CD8⁺ cells/kg, 1.5×10⁵ CD4⁺ and/or CD8⁺ cells/kg,2×10⁵ CD4⁺ and/or CD8⁺ cells/kg, or 1×10⁶ CD4⁺ and/or CD8⁺ cells/kg bodyweight.

In some embodiments, the cells are administered at or within a certainrange of error of, greater than, and/or at least about 1×10⁶, about2.5×10⁶, about 5×10⁶, about 7.5×10⁶, or about 9×10⁶ CD4⁺ cells, and/orat least about 1×10⁶, about 2.5×10⁶, about 5×10⁶, about 7.5×10⁶, orabout 9×10⁶ CD8⁺ cells, and/or at least about 1×10⁶, about 2.5×10⁶,about 5×10⁶, about 7.5×10⁶, or about 9×10⁶ T cells. In some embodiments,the cells are administered at or within a certain range of error ofbetween about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ T cells,between about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ CD4⁺ cells,and/or between about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ CD8⁺cells.

In some embodiments, the cells are administered at or within a toleratedrange of a desired output ratio of multiple cell populations orsub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects,the desired ratio can be a specific ratio or can be a range of ratios.For example, in some embodiments, the desired ratio (e.g., ratio of CD4⁺to CD8⁺ cells) is between at or about 5:1 and at or about 5:1 (orgreater than about 1:5 and less than about 5:1), or between at or about1:3 and at or about 3:1 (or greater than about 1:3 and less than about3:1), such as between at or about 2:1 and at or about 1:5 (or greaterthan about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1,4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In someaspects, the tolerated difference is within about 1%, about 2%, about3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50% of the desired ratio,including any value in between these ranges.

The cell populations and compositions in some embodiments areadministered to a subject using standard administration techniques,including oral, intravenous, intraperitoneal, subcutaneous, pulmonary,transdermal, intramuscular, intranasal, buccal, sublingual, orsuppository administration. In some embodiments, the cell populationsare administered parenterally. The term “parenteral,” as used herein,includes intravenous, intramuscular, subcutaneous, rectal, vaginal, andintraperitoneal administration. In some embodiments, the cellpopulations are administered to a subject using peripheral systemicdelivery by intravenous, intraperitoneal, or subcutaneous injection.

The cell populations obtained using the methods described herein in someembodiments are co-administered with one or more additional therapeuticagents or in connection with another therapeutic intervention, eithersimultaneously or sequentially in any order. In some contexts, the cellsare co-administered with another therapy sufficiently close in time suchthat the cell populations enhance the effect of one or more additionaltherapeutic agents, or vice versa. In some embodiments, the cellpopulations are administered prior to the one or more additionaltherapeutic agents. In some embodiments, the cell populations areadministered after to the one or more additional therapeutic agents.

Following administration of the cells, the biological activity of theengineered cell populations in some embodiments is measured, e.g., byany of a number of known methods. Parameters to assess include specificbinding of an engineered or natural T cell or other immune cell toantigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flowcytometry. In certain embodiments, the ability of the engineered cellsto destroy target cells can be measured using any suitable method knownin the art, such as cytotoxicity assays described in, for example,Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Hermanet al. J. Immunological Methods, 285(1): 25-40 (2004). In certainembodiments, the biological activity of the cells is measured byassaying expression and/or secretion of one or more cytokines, such asCD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity ismeasured by assessing clinical outcome, such as reduction in tumorburden or load.

In certain embodiments, the engineered cells are further modified in anynumber of ways, such that their therapeutic or prophylactic efficacy isincreased. For example, the engineered CAR or TCR expressed by thepopulation can be conjugated either directly or indirectly through alinker to a targeting moiety. The practice of conjugating compounds,e.g., the CAR or TCR, to targeting moieties is known in the art. See,for instance, Wadwa et al., J. Drug Targeting 3: 111 (1995), and U.S.Pat. No. 5,087,616.

IV. DEFINITIONS

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.”

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.”

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

As used herein, “percent (%) amino acid sequence identity” and “percentidentity” when used with respect to an amino acid sequence (referencepolypeptide sequence) is defined as the percentage of amino acidresidues in a candidate sequence (e.g., a streptavidin mutein) that areidentical with the amino acid residues in the reference polypeptidesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters foraligning sequences, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.

An amino acid substitution may include replacement of one amino acid ina polypeptide with another amino acid. Amino acids generally can begrouped according to the following common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative amino acid substitutions will involve exchanging amember of one of these classes for another class.

As used herein, a subject includes any living organism, such as humansand other mammals. Mammals include, but are not limited to, humans, andnon-human animals, including farm animals, sport animals, rodents andpets.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, “depleting” when referring to one or more particularcell type or cell population, refers to decreasing the number orpercentage of the cell type or population, e.g., compared to the totalnumber of cells in or volume of the composition, or relative to othercell types, such as by negative selection based on markers expressed bythe population or cell, or by positive selection based on a marker notpresent on the cell population or cell to be depleted. The term does notrequire complete removal of the cell, cell type, or population from thecomposition.

As used herein, “enriching” when referring to one or more particularcell type or cell population, refers to increasing the number orpercentage of the cell type or population, e.g., compared to the totalnumber of cells in or volume of the composition, or relative to othercell types, such as by positive selection based on markers expressed bythe population or cell, or by negative selection based on a marker notpresent on the cell population or cell to be depleted. The term does notrequire complete removal of other cells, cell type, or populations fromthe composition and does not require that the cells so enriched bepresent at or even near 100% in the enriched composition.

As used herein, the terms “treatment,” “treat,” and “treating,” refer tocomplete or partial amelioration or reduction of a disease or conditionor disorder, or a symptom, adverse effect or outcome, or phenotypeassociated therewith. In certain embodiments, the effect is therapeutic,such that it partially or completely cures a disease or condition oradverse symptom attributable thereto.

As used herein, a “therapeutically effective amount” of a compound orcomposition or combination refers to an amount effective, at dosages andfor periods of time necessary, to achieve a desired therapeutic result,such as for treatment of a disease, condition, or disorder, and/orpharmacokinetic or pharmacodynamic effect of the treatment. Thetherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the subject, and thepopulations of cells administered.

As used herein, a statement that a cell or population of cells is“positive” for a particular marker refers to the detectable presence onor in the cell of a particular marker, typically a surface marker. Whenreferring to a surface marker, the term refers to the presence ofsurface expression as detected by flow cytometry, for example, bystaining with an antibody that specifically binds to the marker anddetecting said antibody, wherein the staining is detectable by flowcytometry at a level substantially above the staining detected carryingout the same procedure with an isotype-matched control or fluorescenceminus one (FMO) gating control under otherwise identical conditionsand/or at a level substantially similar to that for cell known to bepositive for the marker, and/or at a level substantially higher thanthat for a cell known to be negative for the marker.

As used herein, a statement that a cell or population of cells is“negative” for a particular marker refers to the absence of substantialdetectable presence on or in the cell of a particular marker, typicallya surface marker. When referring to a surface marker, the term refers tothe absence of surface expression as detected by flow cytometry, forexample, by staining with an antibody that specifically binds to themarker and detecting said antibody, wherein the staining is not detectedby flow cytometry at a level substantially above the staining detectedcarrying out the same procedure with an isotype-matched control orfluorescence minus one (FMO) gating control under otherwise identicalconditions, and/or at a level substantially lower than that for cellknown to be positive for the marker, and/or at a level substantiallysimilar as compared to that for a cell known to be negative for themarker.

In some embodiments, a decrease in expression of one or markers refersto loss of 1 log¹⁰ in the mean fluorescence intensity and/or decrease ofpercentage of cells that exhibit the marker of at least about 20% of thecells, 25% ofthe cells, 30% of the cells, 35% of the cells, 40% of thecells, 45% of the cells, 50% of the cells, 55% of the cells, 60% of thecells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of thecells, 85% of the cells, 90% of the cell, 95% of the cells, and 100% ofthe cells and any % between 20 and 100% when compared to a referencecell population. In some embodiments, a cell population positive for oneor markers refers to a percentage of cells that exhibit the marker of atleast about 50% of the cells, 55% of the cells, 60% of the cells, 65% ofthe cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% ofthe cells, 90% of the cell, 95% of the cells, and 100% of the cells andany % between 50 and 100% when compared to a reference cell population.

V. EXEMPLARY EMBODIMENTS

Among the embodiments provided herein are:

1. A method for enriching CD4+ or CD8+ T cells, the method including:

(a) performing a first selection in a closed system, said firstselection including enriching for one of (i) CD4+ cells and (ii) CD8+cells from a sample containing primary human T cells, the enrichmentthereby generating a first selected population and a non-selectedpopulation; and

(b) performing a second selection in the closed system, said secondselection including enriching for the other of (i) CD4+ cells and (ii)CD8+ cells from the non-selected population, the enrichment therebygenerating a second selected population,

wherein the method produces an enriched composition, which is enrichedfor CD4+ cells and CD8+ cells and includes cells of the first selectedpopulation and cells of the second selected population.

2. The method of embodiment 1, further including (c) combining cells ofthe first selected population and cells of the second selectedpopulation, thereby producing the enriched composition and/or whereinthe CD4+ and CD8+ cells in the enriched composition are present at aculture-initiating ratio of CD4+ cells to CD8+ cells.

3. The method of embodiment 2, wherein said combining is performed inthe closed system.

4. A method for producing genetically engineered T cells, the methodincluding:

(a) performing a first selection in a closed system, said firstselection including enriching for one of (i) CD4⁺ cells and (ii) CD8+cells from a sample containing primary human T cells, the enrichmentthereby generating a first selected population and a non-selectedpopulation; and

(b) performing a second selection in the closed system, said secondselection including enriching for the other of (i) CD4+ cells and (ii)CD8+ cells from the non-selected population, the enrichment therebygenerating a second selected population;

(c) incubating a culture-initiating composition, which contains cells ofthe first selected population and cells of the second selectedpopulation, in a culture vessel under stimulating conditions, therebygenerating stimulated cells; and

(d) introducing a genetically engineered antigen receptor intostimulated cells generated in (c),

wherein the method thereby generates an output composition includingCD4⁺ T cells and CD8⁺ T cells expressing the genetically engineeredantigen receptor.

5. The method of embodiment 4, further including, prior to step (c),combining cells of the first and second selected cell populations toproduce the culture-initiating composition and/or wherein the CD4+ andCD8+ cells in the culture-initiating composition are present at aculture-initiating ratio of CD4+ cells to CD8+ cells.

6. The method of embodiment 5, wherein said combining is performed inthe closed system.

7. The method of any of embodiments 1-6, wherein one or more of thesteps are carried out in an automated fashion and/or wherein the closedsystem is automated.

8. The method of any of embodiments 2-7, wherein the culture-initiatingratio of CD4+ to CD8⁺ cells is between at or about 10:1 and at or about1:10, between at or about 5:1 and at or about 1:5, or between at orabout 2:1 and at or about 1:2.

9. The method of any of embodiments 2-8, wherein the culture-initiatingratio of CD4+ to CD8+ cells is at or about 1:1.

10. The method of any of embodiments 2-6, wherein the sample is obtainedfrom a human subject and:

the culture-initiating ratio of CD4⁺ to CD8⁺ cells is different than theratio of CD4⁺ to CD8⁺ cells in the sample from the subject; and/or

the culture-initiating ratio of CD4⁺ to CD8⁺ cells is at least 10%, atleast 20%, at least 30%, at least 40%, or at least 50% greater or lessthan the ratio of CD4⁺ to CD8⁺ cells in the sample from the subject.

11. The method of any of embodiments 1-10, wherein enriching cells inthe first and/or second selection includes performing positive selectionor negative selection based on expression of a cell surface marker.

12. The method of embodiment 11, wherein enriching for cells in thefirst and/or second selection includes negative selection, whichincludes depleting cells expressing a non-T cell surface marker.

13. The method of embodiment 12, wherein the non-T cell marker comprisesCD14.

14. The method of any of embodiments 1-13, wherein enriching cells inthe first or second selection includes performing a plurality ofpositive or negative selection steps based on expression of a cellsurface marker or markers to enrich for CD4+ or CD8+ cells.

15. The method of any of embodiments 1-14, wherein the enriching cellsin the first and/or second selection includes immunoaffinity-basedselection.

16. The method of embodiment 15, wherein the immununoaffinity-basedselection is effected by contacting cells with an antibody capable ofspecifically binding to a cell surface marker and recovering cells boundto the antibody, thereby effecting positive selection, or recoveringcells not bound to the antibody, thereby effecting negative selection,wherein the recovered cells are enriched for the CD4+ cells or the CD8+cells and antibody is immobilized on a magnetic particle.

17. The method of any of embodiments 1-13, wherein the first selectionand second selection are carried out in separate separation vessels,which are operably connected.

18. The method of embodiment 17, wherein the separation vessels areoperably connected by tubing.

19. The method of any of embodiments 15-18, wherein theimmunoaffinity-based selection is effected by contacting cells with anantibody immobilized on or attached to an affinity chromatographymatrix, said antibody capable of specifically binding to a cell surfacemarker to effect positive or negative selection of CD4+ or CD8+ cells.

20. The method of embodiment 19, wherein:

the antibody further includes one or more binding partners capable offorming a reversible bond with a binding reagent immobilized on thematrix, whereby the antibody is reversibly bound to said matrix duringsaid contacting; and

cells expressing a cell surface marker specifically bound by theantibody on said matrix are capable of being recovered from the matrixby disruption of the reversible binding between the binding reagent andbinding partner.

21. The method of embodiment 17, wherein:

the binding partner is selected from among biotin, a biotin analog, anda peptide capable of binding to the binding reagent; and

the binding reagent is selected from among streptavidin, a streptavidinanalog or mutein, avidin and an avidin analog or mutein.

22. The method of embodiment 21, wherein:

the binding partner includes a sequence of amino acids set forth in SEQID NO:6; and/or

the binding reagent is a streptavidin mutein including the sequence ofamino acids set forth in SEQ ID NO: 12, 13, 15 or 16.

23. The method of any of embodiments 19-21, further including, aftercontacting cells in the sample to an affinity chromatography matrix inthe first selection and/or second selection, applying a competitionreagent to disrupt the bond between the binding partner and bindingreagent, thereby recovering the selected cells from the matrix.

24. The method of embodiment 23, wherein the competition reagent isbiotin or a biotin analog.

25. The method of any of embodiments 20-24, wherein the antibody orantibodies in the first and/or second selection has a dissociation rateconstant (k_(off)) for binding and the cell surface marker of greaterthan or greater than about 3×10⁻⁵ sec⁻¹.

26. The method of any of embodiments 20-25, wherein the antibody orantibodies in the first and/or second selection has an affinity for thecell surface marker of a dissociation constant (K_(d)) in the range ofabout 10⁻³ to 10⁻⁷ or in the range of about 10⁻⁷ to about 10⁻¹⁰.

27. The method of any of embodiments 20-26, wherein the chromatographymatrix of the first and/or second selection is packed in a separationvessel, which is a column.

28. The method of embodiment 19-27, wherein the affinity chromatographymatrix adsorbs and/or is capable of selecting at least or at least about50×10⁶ cells/mL, 100×10⁶ cells/mL, 200×10⁶ cells/mL or 400×10⁶ cells/mL.

29. The method of any of embodiments 19-28, wherein the first and thesecond selection steps comprise the use of the affinity chromatographymatrix and the matrix used in the first and second selection steps areat sufficient relative amounts to achieve the culture-initiation ratio.

30. The method of any of embodiments 1-29, wherein the enriching for theCD4+ cells includes positive selection based on surface expression ofCD4.

31. The method of any of embodiments 1-29, wherein the enriching for theCD8+ cells includes positive selection based on surface expression ofCD8.

32. The method of any of embodiments 1-29, wherein the one of the firstand second selections that includes enriching for the CD8+ cells furtherincludes enriching for central memory T (T_(CM)) cells; and/or

enriching for cells expressing a marker selected from among CD28, CD62L,CCR7, CD127 and CD27.

33. The method of any of embodiments 1-32, wherein:

the first selection includes enriching for the CD8+ cells and the secondselection includes enriching for the CD4+ cells; and

the first selection further includes enriching for central memory T(T_(CM)) cells and/or enriching for cells expressing a marker selectedfrom among CD28, CD62L, CCR7, CD127 and CD27.

34. The method of embodiment 32 or embodiment 33 or embodiment 35,wherein enriching for central memory T (T_(CM)) cells and/or enrichingfor cells expressing the marker includes:

selecting from the first and/or second selected cell population, whichis enriched for CD8⁺ cells, cells expressing CD62L; and/or

selecting, from the first and/or second selected cell population, whichis enriched for CD8⁺ cells, cells expressing CD27; and/or

selecting, from the first and/or second selected cell population, whichis enriched for CD8⁺ cells, cells expressing CCR7; and/or

selecting, from the first and/or second selected cell population, whichis enriched for CD8⁺ cells, cells expressing CD28; and/or

selecting, from the first and/or second selected cell population, whichis enriched for CD8⁺ cells, cells expressing CD127.

35. The method of any of embodiments 1-32, wherein:

the first selection includes enriching for the CD4+ cells and the secondselection includes enriching for the CD8+ cells; and

the second selection further includes enriching for central memory T(T_(CM)) cells.

36. The method of embodiment 35, wherein:

(i) the first selection includes enriching CD4+ cells by positiveselection based on surface expression of CD4, thereby generating thefirst selected population, which is enriched for CD4+ primary human Tcells, and the non-selected sample;

(ii) the second selection includes enriching for the CD8+ cells andfurther includes enriching the second selected sample for central memoryT (T_(CM)) cells, wherein enriching for central memory T (T_(CM)) cellsincludes:

-   -   negative selection to deplete cells expressing a surface marker        present on nave T cells and positive selection for cells        expressing a surface marker present on central memory T (T_(CM))        cells and not present on another memory T cell sub-population;        or    -   positive selection for cells expressing a surface marker present        on central memory T cells and not present on nave T cells and        positive selection for cells expressing a surface marker present        on central memory T (T_(CM)) cells and not present on another        memory T cell sub-population;    -   thereby generating CD8+ primary human T cells enriched for        T_(CM) cells.

37. The method of embodiment 36, wherein:

the marker present on nave T cells comprises CD45RA; and

enriching for central memory T (T_(CM)) cells includes negativeselection to deplete cells expressing CD45RA and positive selection forcells expressing a surface marker present on central memory T (T_(CM))cells and not present on another memory T cell sub-population.

38. The method of embodiment 37, wherein:

the surface marker present on central memory T cells and not present onnave T cells comprises CD45RO;

the enrichment for central memory T (T_(CM)) cells includes positiveselection for cells expressing CD45RO and positive selection for cellsexpressing surface marker present on central memory T (T_(CM)) cells andnot present on another memory T cell sub-population.

39. The method of any of embodiments 36-38, wherein the surface markerpresent on central memory T (T_(CM)) cells and not present on anothermemory T cell sub-population is selected from the group consisting ofCD62L, CCR7, CD27, CD127, and CD44.

40. The method of embodiment 39, wherein the surface marker present oncentral memory T (T_(CM)) cells and not present on another memory T cellsub-population is CD62L.

41. The method of any of embodiments 32-40, wherein the CD8⁺ populationin the enriched composition or the culture-initiating compositionincludes at least 50% central memory T (T_(CM)) cells or includes lessthan 20% nave T (T_(N)) cells or includes at least 80% CD62L+ cells.

42. A method for enriching CD4+ and CD8+ T cells, the method includingcontacting cells of a sample containing primary human T cells with afirst immunoaffinity reagent that specifically binds to CD4 and a secondimmunoaffinity reagent that specifically binds to CD8 in an incubationcomposition, under conditions whereby the immunoaffinity reagentsspecifically bind to CD4 and CD8 molecules, respectively, on the surfaceof cells in the sample; and

recovering cells bound to the first and/or the second immunoaffinityreagent, thereby generating an enriched composition including CD4+ cellsand CD8+ cells at a culture-initiating ratio, wherein:

the first and/or second immunoaffinity reagent are present in theincubation composition at a sub-optimal yield concentration, whereby theenriched composition contains less than 70% of the total CD4+ cells inthe incubation composition and/or less than 70% of the CD8+ cells in theincubation composition, thereby producing a composition enriched forCD4+ and CD8+ T cells.

43. A method for producing genetically engineered T cells, the methodincluding:

(a) enriching for primary human T cells from a sample containing primaryhuman T cells including:

-   -   contacting cells of said sample with a first immunoaffinity        reagent that specifically binds to CD4 and a second        immunoaffinity reagent that specifically binds to CD8 in an        incubation composition, under conditions whereby the        immunoaffinity reagents specifically bind to CD4 and CD8        molecules, respectively, on the surface of cells in the sample;        and    -   recovering cells bound to the first and/or the second        immunoaffinity reagent, thereby generating an enriched        composition including CD4+ cells and CD8+ cells at a        culture-initiating ratio, wherein:    -   the first and/or second immunoaffinity reagent are present in        the incubation composition at a sub-optimal yield concentration,        whereby the enriched composition contains less than 70% of the        total CD4+ cells in the incubation composition and/or less than        70% of the CD8+ cells in the incubation composition; and

(b) incubating cells of the enriched composition in a culture-initiationcomposition in a culture vessel under stimulating conditions, therebygenerating stimulated cells, wherein the cells are at or substantiallyat the culture-initiating ratio; and

(c) introducing a genetically engineered antigen receptor intostimulated cells of (b), thereby generating an output compositionincluding CD4⁺ T cells and CD8⁺ T cells expressing the geneticallyengineered antigen receptor.

44. The method of embodiment 42 or embodiment 43, wherein enriching forprimary human T cells is performed in a closed system.

45. The method of any of embodiments 42-44, wherein the first and secondimmunoaffinity reagent are present in the incubation composition at asub-optimal yield concentration, whereby the enriched compositioncontains less than 70% of the total CD4+ cells in the incubationcomposition and less than 70% of the total CD8+ cells in the incubationcomposition.

46. The method of any of embodiments 42-45, wherein:

the first immunoaffinity reagent is present in the incubationcomposition at a sub-optimal yield concentration, whereby the enrichedcomposition contains less than 60%, less than 50%, less than 40%, lessthan 30% or less than 20% of the total CD4+ cells in the incubationcomposition; and/or

the second immunoaffinity reagent is present in the incubationcomposition at a sub-optimal yield concentration, whereby the enrichedcomposition contains less than 60%, less than 50%, less than 40%, lessthan 30% or less than 20% of the total CD8⁺ cells in the incubationcomposition.

47. The method of embodiment any of embodiments 42-46, wherein thesample contains at least 1×10⁹ CD3+ T cells.

48. The method of any of embodiments 42-47, wherein the concentration ofone of the first and second immunoaffinity reagent in the incubationcomposition is greater than that of the other, whereby the greaterconcentration effects a higher yield in the enriched composition of CD4+cells or CD8+ cells, respectively, compared to the yield of the other ofthe CD4+ or CD8+ cells, thereby producing the culture-initiating ratioin the enriched composition.

49. The method of embodiment 48, wherein:

the concentration of one of the first and second immunoaffinity reagentsis greater, as compared to the concentration of the other of the firstand second immunoaffinity reagents, by at least 1.2-fold, 1.4-fold,1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold,7.0-fold, 8.0-fold, 9.0-fold or 10-fold; and/or

the higher yield in the enriched concentration is greater by 1.2-fold,1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold,6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold or 10-fold.

50. The method of any of embodiments 42-49, wherein theculture-initiating ratio of CD4⁺ to CD8⁺ cells is between at or about10:1 and at or about 1:10, between at or about 5:1 and at or about 1:5or is between at or about 2:1 and at or about 1:2.

51. The method of any of embodiments 42-50, wherein theculture-initiating ratio of CD4+ to CD8+ cells is at or about 1:1.

52. The method of any of embodiments 42-51, wherein:

the culture-initiating ratio of CD4⁺ to CD8⁺ cells is different than theratio of CD4⁺ to CD8⁺ cells in the sample from the subject; and/or

the culture-initiating ratio of CD4⁺ to CD8⁺ cells is at least 10%, atleast 20%, at least 30%, at least 40%, or at least 50% greater or lessthan the ratio of CD4⁺ to CD8⁺ cells in the sample from the subject.

53. The method of any of embodiments 42-52, wherein greater than 95% orgreater than 98% of the cells in the culture-initiation composition areCD4+ cells and CD8+ cells.

54. The method of any of embodiments 42-53, wherein each of theimmunoaffinity reagents contains an antibody.

55. The method of embodiment 54, wherein the antibody is immobilized onthe outside surface of a sphere.

56. The method of embodiment 55, wherein the sphere is a magnetic bead.

57. The method of embodiment 55 or embodiment 56, wherein:

the antibody contains one or more binding partners capable of forming areversible bond with a binding reagent immobilized on the sphere,whereby the antibody is reversibly immobilized to said sphere; and

the method further includes after contacting cells in the sample to thefirst and second immunoaffinity reagent, applying a competition reagentto disrupt the bond between the binding partner and binding reagent,thereby recovering the selected cells from the sphere.

58. The method of embodiment 57, wherein:

the binding partner is selected from among biotin, a biotin analog, or apeptide capable of binding to the binding reagent; and

the binding reagent is selected from among streptavidin, a streptavidinanalog or mutein, avidin, an avidin analog or mutein.

59. The method of embodiment 58, wherein:

the binding partner contains a peptide including the sequence of aminoacids set forth in SEQ ID NO:6 and/or

the binding reagent is streptavidin mutein including the sequence ofamino acids set forth in SEQ ID NO: 12, 13, 15 or 16.

60. The method of embodiment 58 or embodiment 59, wherein:

the binding reagent is a multimer including one or more monomers ofstreptavidin or a streptavidin mutein; and

the binding partner is a peptide including a sequential arrangement ofat least two modules each capable of reversibly binding with at leastone monomer of the binding reagent.

61. The method of embodiment 60, wherein the binding partner containsthe sequence of amino acids set forth in any of SEQ ID NOS: 7-10.

62. The method of any of embodiments 57-61, wherein the competitionreagent is biotin or a biotin analog.

63. The method of any of embodiments 42-63, wherein the antibody orantibodies in the first and/or second selection has a dissociation rateconstant (k_(off)) for binding between the antibody and cell surfacemarker of greater than or greater than about 3×10⁻⁵ sec⁻¹.

64. The method of any of embodiments 42-63, wherein the antibody orantibodies in the first and/or second selection has an affinity with adissociation constant (K_(d)) in the range of about 10⁻³ to 10⁻⁷ or witha dissociation constant in the range of about 10⁻⁷ to about 10⁻¹⁰.

65. The method of any of embodiments 42-63, wherein one or more stepsare carried out in an automated fashion and/or wherein the closed systemis automated.

66. The method of any of embodiments 2, 3 and 5-41, including:

prior to performing the first selection and/or second selection,determining the ratio of CD4+ to CD8+ T cells in the sample; and

based on the ratio of CD4+ to CD8+ T cells in the sample, adjusting thefirst and/or second selection to produce the composition including CD4+cells and CD8+ cells at the culture-initiating ratio.

67. The method of embodiment 66, wherein:

the first and/or second selection includes immunoaffinity-basedselection including an affinity chromatography matrix; and

adjusting the first and/or second selection includes choosing the amountof affinity chromatography matrix in the first and/or second selectionsufficient to achieve the culture-initiation ratio.

68. The method of any of embodiments 42-65, including:

prior to contacting cells of the sample with the first and secondimmunoaffinity reagent, determining the ratio of CD4+ to CD8+ T cells inthe sample; and

based on the ratio of CD4+ to CD8+ T cells in the sample, choosing theconcentration of the first and/or second immunoaffinity reagent toproduce the enriched composition including CD4+ cells and CD8+ cells atthe culture-initiating ratio.

69. The method of any of embodiments 1-68, wherein the method results anoutput composition including a ratio of CD4⁺ to CD8⁺ cells that isbetween at or about 2:1 and at or about 1:5.

70. The method of embodiment 69, wherein the ratio of CD4⁺ to CD8⁺ cellsin the output composition is 1:1 or is about 1:1.

71. The method of any of embodiments 1-70, wherein the sample isobtained from a subject.

72. The method of embodiment 71, wherein the subject is a subject towhom said genetically engineered T cells or cells for adoptive celltherapy will be administered.

73. The method of embodiment 72, wherein the subject is a subject otherthan a subject to whom said genetically engineered T cells or cells foradoptive therapy will be administered.

74. The method of any of embodiments 1-73, wherein the sample is bloodor a blood-derived sample.

75. The method of any of embodiments 1-74, wherein the sample is a whiteblood cell sample.

76. The method of any of embodiments 1-75, wherein the sample is anapheresis, peripheral blood mononuclear cell (PBMC), or leukapheresissample.

77. The method of any of embodiments 4-41 or 43-76, wherein incubatingthe composition in a culture vessel under stimulating conditions isperformed prior to, during and/or subsequent to introducing agenetically engineered antigen receptor.

78. The method of any of embodiments 4-41 or 43-76, wherein incubatingthe composition in a culture vessel under stimulating conditions isperformed prior to, during and subsequent to introducing a geneticallyengineered antigen receptor.

79. The method of any of embodiments 4-41 or 43-78, wherein thestimulating conditions comprise conditions whereby T cells of thecomposition proliferate.

80. The method of any of embodiments 4-41 or 43-79, wherein thestimulating condition includes an agent capable of activating one ormore intracellular signaling domains of one or more components of a TCRcomplex.

81. The method of embodiment 80, wherein the one or more components ofthe TCR complex contains the CD3 zeta chain.

82. The method of any of embodiments 4-41 or 43-81, wherein thestimulating condition contains the presence of an anti-CD3 antibody, andanti-CD28 antibody, anti-4-1BB antibody, and/or a cytokine.

83. The method of embodiment 82, wherein the anti-CD3 antibody and/orthe anti-CD28 antibody is present on the surface of a solid support.

84. The method of embodiment 83, wherein the cytokine includes IL-2,IL-15, IL-7, and/or IL-21.

85. The method of any of embodiments 4-41 or 43-84, wherein thegenetically engineered antigen receptor includes a T cell receptor (TCR)or a functional non-TCR antigen receptor.

86. The method of embodiment 85, wherein the receptor specifically bindsto an antigen expressed by cells of a disease or condition to betreated.

87. The method of any of embodiment 85 or 86, wherein the antigenreceptor is a chimeric antigen receptor (CAR).

88. The method of embodiment 87, wherein the CAR contains anextracellular antigen-recognition domain and an intracellular signalingdomain including an ITAM-containing sequence and an intracellularsignaling domain of a T cell costimulatory molecule.

89. A method of treatment, said method including:

(a) producing an output composition including CD4⁺ T cells and CD8⁺ Tcells according to any of embodiments 1-88; and

(b) administering cells of the output composition to a subject.

90. The method of embodiment 89, wherein the sample from which the cellsare isolated is derived from the subject to which the cells areadministered.

91. A composition of cells produced by the method of any of embodiments1-88.

92. The composition of embodiment 91, including a pharmaceuticallyacceptable carrier.

93. A method of treatment, said method including administering to asubject a composition of cells of embodiment 91 or 92.

94. The method of embodiment 93, wherein the genetically engineeredantigen receptor specifically binds to an antigen associated with thedisease or condition.

95. The method of treatment of embodiment 94, wherein the disease orcondition is a cancer.

96. The composition of embodiment 91 or embodiment 92 for use intreating a disease or condition in a subject.

97. Use of a composition of embodiment 91 or embodiment 92 for themanufacture of a medicament for treating a disease or disorder in asubject.

98. The composition of embodiment 96 or use of embodiment 97, whereinthe genetically engineered antigen receptor specifically binds to anantigen associated with the disease or condition.

99. The composition or use of any of embodiments 96-98, wherein thedisease or condition is a cancer.

100. A closed apparatus system for purification of target cells,including:

a) a first affinity chromatography matrix including a first bindingagent immobilized thereon, which binding agent specifically binds to afirst cell surface marker present on a first cell, wherein the firstaffinity chromatography matrix is operably connected to a storagereservoir including a cell sample via a first operable connection, saidfirst operable connection capable of permitting passage of cells fromthe storage reservoir to the first affinity chromatography matrix andwherein the first affinity chromatography matrix is operably connectedto an output vessel via a second operable connection; and

b) a second affinity chromatography matrix including a second bindingagent immobilized thereon, which second binding agent specifically bindsto a second cell surface marker present on a second cell, wherein thefirst affinity chromatography matrix is operably connected via a thirdoperable connection to the second affinity chromatography matrix, saidthird operable connection capable of permitting passage of cells havingpassed through the first affinity chromatography matrix and not bound tothe first binding agent to the second affinity chromatography matrix,and the second affinity chromatography matrix is operably connected viaa fourth operable connection to the output vessel, the fourth operableconnection capable of permitting passage of cells having bound to andbeen eluted from the first and/or second affinity chromatography matrix,and the second affinity chromatography matrix is operably linked, via afifth operable connection, to a waste vessel, the fifth operableconnection capable of permitting passage of cells having passed throughthe first affinity chromatography matrix and not bound to the firstbinding agent and having passed through the second affinitychromatography matrix and not bound to the second binding agent;

c) the output vessel; and

d) the waste vessel,

wherein the system is configured to permit, within the closed system,collection of, in the output vessel in a single composition, (i) cellshaving bound and been recovered from the first affinity chromatographymatrix and (ii) cells having passed through and not bound to the firstaffinity chromatography matrix and having bound and been recovered fromthe second chromatography matrix.

101. The closed apparatus of embodiment 100, wherein one or more of saidoperable connections contains tubing connecting the storage reservoir,first affinity chromatography matrix, second affinity chromatographymatrix and/or culture vessel.

102. The closed apparatus of embodiment 101, wherein the tubing isconnected to a stopcock, valve or clamp.

103. The closed apparatus system of any of embodiments 100-102, wherein:

(a) the first affinity chromatography matrix is one of a CD4+ affinitychromatography matrix or a CD8+ affinity chromatography matrix; and

(b) the second affinity chromatography matrix is the other of the CD4+affinity chromatography matrix or the CD8+ affinity chromatographymatrix.

104. The closed apparatus system of any of embodiments 100-103, furtherincluding:

d) a third affinity chromatography matrix including a third bindingagent immobilized thereon, which binding agent specifically binds to athird cell surface marker, whereby the third binding agent is capable ofbinding cells expressing the third cell surface marker, wherein thethird operable connection further operably connects the third affinitychromatography matrix to the first matrix and a sixth operableconnection operably connects the third affinity chromatography matrix tothe output container, such that the sixth operable connection is capableof permitting passage of cells having bound to and been recovered fromthe first matrix and having bound to and been recovered from the thirdmatrix to the output container, and

the fifth operable connection further operably connects the thirdaffinity chromatography matrix to the waste container, such that thefifth operable connection is capable of permitting cells having passedthrough and not bound to the third column to the waste container.

105. The closed apparatus system of any of embodiments 100-103, furtherincluding:

d) a third affinity chromatography matrix including a third bindingagent immobilized thereon, which binding agent specifically binds to athird cell surface marker, whereby the third affinity chromatographymatrix is capable of binding cells expressing the third cell surfacemarker, wherein

the second operable connection further operably connects the thirdaffinity chromatography matrix to the first matrix and to the outputcontainer, such that the second operable connection is capable ofpermitting cells having bound to and been recovered from the firstmatrix and having bound to and been recovered from the third matrix tothe output container.

106. The closed apparatus system of embodiment 104 or 105, wherein:

the first affinity chromatography matrix includes a biding agent thatspecifically binds CD8;

the second affinity chromatography matrix includes a binding agent thatspecifically binds CD4; and

the third affinity chromatography matrix includes a binding agent thatspecifically binds a marker expressed on central memory T (T_(CM))cells.

107. The closed apparatus system of embodiment 106, wherein the bindingagent of the third affinity chromatography matrix selectively binds to acell surface marker selected from among CD62L, CD45RA, CD45RO, CCR7,CD27, CD127, and CD44.

108. The closed apparatus system of any of embodiments 100-107, whereinone or more or all of the affinity chromatography matrices are furtheroperably connected to an elution buffer reservoir including one or morecompetition reagents.

109. The closed apparatus system of any of embodiments 100-107, whereinthe competition reagent is one or more of the group consisting ofbiotin, a biotin analog, and a peptide capable of binding to thechromatography matrix.

110. The closed apparatus system of any of embodiments 100-107, whereinone or more or all of the third, fourth, or sixth operable connectionsfurther includes a competition agent removal chamber.

111. The closed apparatus system of embodiment 110, wherein thecompetition agent removal chamber further contains a binding reagent.

112. The closed apparatus system of embodiment 111, wherein the bindingreagent includes one or more of the group consisting of streptavidin, astreptavidin analog or mutein, avidin and an avidin analog or mutein.

113. The closed apparatus system of any of embodiments 100-112, whereinone or more or all of the binding agents is an antibody.

114. The closed apparatus system of any of embodiments 100-112, whereinthe one or more or all of the binding agents is reversibly bound to theaffinity chromatography matrix.

VI. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Generation of a CD4+ and CD8+ T Cell Composition Using aSingle Process Stream by Immunomagnetic Separation for GeneticEngineering and Adoptive Cell Therapy

In an exemplary method, a CD4⁺ T cell population and a CD8⁺ T cellpopulation, enriched for central memory T cells, are isolated from anapheresis product sample, and subsequently incubated and engineered,followed by administration to a subject. The isolation procedure iscarried out using a single process stream by immunomagnetic separationusing the CliniMACS® Prodigy system. The process is streamlined ascompared to other methods, in which CD4⁺ cells are separated from afirst apheresis fraction and CD8⁺ cells are separated and furtherdepleted/enriched, from a second apheresis fraction, using a CliniMACS®Prodigy device and three separate tubing sets.

The streamlined process is performed using one tubing set for isolationof the CD4⁺ and CD8⁺ cell populations, without transferring the cellpopulations from one vessel (e.g., tubing set) to another.

The CD4⁺ cell population is isolated by incubating an apheresis samplewith a CliniMACS® CD4 reagent. The cells are then separated by theCliniMACS® Prodigy device set to run an enrichment program, and bothcell fractions (i.e., the immunomagnetically selected enriched CD4⁺ cellfraction and the flow-through cell fraction) are retained. The enriched(positive) fraction is an isolated CD4⁺ T cell population. A populationof CD8⁺ T cells is isolated by incubating the flow-through (negative)fraction with a CliniMACS® CD14 reagent and a CliniMACS® CD45RA reagentor CD19 reagent. The CliniMACS® Prodigy device is set to run a depletionprogram. The cell/reagent mixture then is separated by the CliniMACS®Prodigy device using the same tubing set used in the first separationstep. The flow-through (negative) fraction from which CD14⁺/CD45RA⁺ orCD14+/CD19+ cells have been depleted is incubated with CliniMACS® CD62Lreagent. The cell/reagent mixture is separated using the CliniMACS®Prodigy device running an enrichment program using the same tubing set.The positive fraction is an isolated CD8⁺ cell population enriched forcentral memory cells.

The isolated CD4+ and CD8+ populations are combined in the same culturevessel in a culture-initiating composition at a culture initiationratio. The culture-initiation ratio is designed to achieve a particulardesired output ratio, or a ratio that is within a certain range oftolerated error of such a desired output ratio, following the incubationand/or engineering steps, or designed to do so a certain percentage ofthe time. The cells are incubated under stimulating conditions, such asusing anti-CD3/anti-CD28 beads in the presence IL-2 (100 IU/mL) for 72hours at 37° C., within the Prodigy system.

The cell composition is optionally periodically assessed and/or adjustedat one or more times subsequent to the initiation of the incubation orculture, such as at a time during the incubation. Assessment includesmeasuring proliferation rate, measuring degree of survival, determiningphenotype, e.g., expression of one or more surface or intracellularmarkers, such as proteins or polynucleotides, and/or adjusting thecomposition or vessel for temperature, media component(s), oxygen orcarbon dioxide content, and/or presence or absence or amount or relativeamount of one or more factors, agents, components, and/or cell types,including subtypes.

Cells so-incubated then are genetically engineered, by introducing intothe cells recombinant genes for expression of recombinant antigenreceptors, such as chimeric antigen receptors (CARs) or recombinantTCRs. The introduction is performed in the contained environment withinthe CliniMACS® Prodigy device, with CD4⁺ and CD8⁺ cells present in thesame composition and vessel. The method results in an output compositionwith engineered CD4⁺ and CD8⁺ T cells.

Example 2 Generation of a CD4⁺ and CD8⁺ T Cell Composition by SequentialPurification in a Closed System

This example demonstrates an exemplary method of enriching or selectingfor a CD4+ T cell population and a CD8+ T cell population from anapheresis product sample obtained from a subject. The process isperformed using a closed system with multiple chromatography columns forsequential positive selection of the CD4+ and CD8+ T cell populationsfrom the same starting sample.

A. Enrichment for CD4+ T Cell Population and CD8+ T Cell Population

In the exemplary process, depicted in FIG. 1A, a series ofimmunochromatographic selection columns and removal columns are arrangedin a closed apparatus 14 operably connected to each other and aperistaltic pump 8 through various tubing lines and valves 13 to controlflow of liquid phase.

The first selection column 1 contains a chosen volume of an affinitychromatography matrix 3, such as an agarose resin, such as a resin asdescribed in published U.S. Patent Appl. No. US2015/0024411 for theisolation of T cells, such as one with an exclusion limit designed to begreater than the size of a T cell, such as an agarose as obtained fromAgarose Beads Technologies, Madrid, Spain, with a reduced exclusion sizecompared to Superflow™ Agarose, which has an exclusion size of 6×10⁶Daltons. The matrix in the first column is bound to multimers ofStrep-Tactin® (e.g. containing a streptavidin mutant set forth in any ofSEQ ID NO:12, 13, 15 or 16, IBA GmbH, Germany or as described inInternational Published PCT Appl. Nos. WO 2014/076277). The secondselection column 2 contains a chosen volume of an affinitychromatography matrix 4, such as an agarose resin, such as a resin asdescribed above, which is also bound to multimers of Strep-Tactin®.

A reservoir containing an anti-CD8 Fab fragment 18 is loaded to runthrough the pump 8, tubing, and valves 13 such that the anti-CD8 Fab isapplied to the first selection column 1, whereby the anti-CD8 Fabbecomes immobilized on the Strep-Tactin® on the affinity matrix 3 with aTwin Strep-Tag® (e.g. set forth in SEQ ID NO: 10; IBA GmbH) in the FabFragment, which is fused to the carboxy-terminus of its heavy chain (seee.g. published U.S. Patent Appl. No. US2015/0024411). In someembodiments, a washing buffer from a washing buffer reservoir 6, such asphosphate buffered saline (PBS) containing 0.5% bovine serum albumin,human serum albumin, or recombinant human serum albumin, is loaded torun through the first selection column via operably coupled tubing. Theflow-through is directed to the waste container 10 via valves 13operably connecting the first selection column and waste container. Insome embodiments, the washing step is repeated a plurality of times.

A reservoir containing an anti-CD4 Fab fragment 19 is loaded to runthrough the pump 8, tubing, and valves 13 such that the anti-CD4 Fab isapplied to the second selection column 2, whereby the anti-CD8 Fabbecomes immobilized on the Strep-Tactin® on the affinity matrix 4 with aTwin Strep-Tag®. In some embodiments, a washing buffer from a washingbuffer reservoir 6 is run through the second selection column viaoperably coupled tubing. The flow-through is directed to the wastecontainer 10 via valves 13 operably connecting the second selectioncolumn and waste container. In some embodiments, the washing step isrepeated a plurality of times.

The volume of affinity matrix reagent contained in the first selectioncolumn and the second selection column can be the same or different, andcan be chosen based on the desired yield of the selection and/or desiredratio of CD4+ cells to CD8+ cells following selection. The volume sochosen is based on an assumption of an average yield of 1×10⁸ cellsbeing capable of selection per each 1 mL volume of filled column. In oneexemplary process, the column volumes are the same, for example, using a2 mL volume for the column for the anti-CD8 Fab fragment and a 2 mLcolumn for the anti-CD4 Fab fragment. The column length and/or columndiameter can be chosen to fit the desired volume, for example, toachieve a desired culture-initiation ratio of CD4⁺ cells to CD8+ cells.In some embodiments, to accommodate the volume of affinity matrix, aplurality of columns having immobilized thereto the same Fab reagent canbe added directly in series and operably connected to each other via atubing line.

To achieve selection of cells, an apheresis sample is applied to thefirst selection column 1, whereby CD8+ T cells, if present in thesample, remain bound to the resin 3 of the first column, as unselectedcells (containing CD8− cells) pass through the column. The number ofcells in the starting sample used in some examples is chosen to be abovethe number of cells to be selected based on the combined volume of thematrices, such as an amount greater than the capacity of the column foreach selection (e.g. an amount having greater than 200 million CD8+cells and greater than 200 million CD4+ cells, e.g., greater by at leastabout 10% or 20% or greater). The flow-through containing unselectedcells (negative fraction) then passes from the first column to a secondselection column 2 via operably coupled tubing. CD4+ T cells remainbound to the resin 4 in the second column. The flow through containingfluid and further unselected cells (negative cells) is directed to awaste container 10 via valves 13 operably connecting the secondselection column and waste container.

In some embodiments, the first selection column can alternatively be ananti-CD4 affinity chromatography matrix and the second selection columncan be an anti-CD8 affinity chromatography matrix.

From a washing buffer reservoir 6, a washing buffer is run through thefirst column and the second column via operably coupled tubing. Theflow-through containing any cells that are washed from the first andsecond columns is directed to the waste container 10 via valves 13operably connecting the second selection column and waste container. Insome embodiments, the washing step is repeated a plurality of times.

From an elution buffer reservoir 7, a buffer containing an eluent, suchas low concentrations of biotin or an analog thereof, for example 2.5 mMdesthiobiotin, is loaded to run through the first column and the secondcolumn via operably coupled tubing. In some embodiments, the eluentcontains cell culture media. The flow-through, containing enriched CD4+and CD8+ cells and residual biotin or analog, is directed to a removalchamber 9 to remove biotin or an analog thereof. The removal chamber 9is a column of Superflow™ Sepharose® beads having Strep-Tactin®immobilized thereto with a volume sufficient to remove biotin or abiotin analog from the sample, such as a column having a bed volume of 6mL with a binding capacity of 300 nanomol biotin/mL. The flow-throughcontaining enriched cells positively selected for CD4+ and CD8+ isdirected to a culture vessel 12, such as a bag, via valves operablyconnecting the removal chamber 9 and culture vessel. In someembodiments, the elution step is repeated.

In some embodiments, after performing the elution step, an activationbuffer containing a T-cell activation reagent (e.g. anti-CD3, anti-CD28,IL-2, IL-15, IL-7, and/or IL-21) replaces the wash buffer and isdirected through the removal chamber and flow through directed to theculture vessel. The positive fraction collected in the culture vessel isan isolated and combined CD4+ and CD8+ cell population.

B. Enrichment for a CD4+ T Cell Population and a CD8+ T Cell Population,Enriched for Cells Expressing a Marker on Central Memory T (T_(CM))Cells

In the exemplary process, depicted in FIG. 1B, a series ofimmunochromatographic selection columns and removal columns are arrangedin a closed apparatus 14 operably connected to each other and aperistaltic pump 8 through various tubing lines and valves 13 to controlflow of liquid phase.

The first selection column 1 and second selection column 2 are the sameas described above with respect to Example 2A. In addition, the processfurther includes a third selection column 15 that contains a chosenvolume of an affinity matrix 17, such as an agarose resin, such as aresin as described in published U.S. Patent Appl. No. US2015/0024411 forthe isolation of T cells, such as one as described above in Example 2A.

The anti-CD8 Fab and the anti-CD4 Fab are applied to the first columnand second column, respectively, as described in Example 2A. A reservoircontaining a further Fab fragment against a marker expressed on centralmemory T (T_(CM)) cells 20, such as one of CD28, CD62L, CCR7, CD27 orCD127, is loaded to run through the pump 8, tubing, and valves 13 suchthat the further Fab is applied to the third selection column 15,whereby the further Fab becomes conjugated to the Strep-Tactin® on theaffinity matrix 17 with a Twin Strep-Tag® (SEQ ID NO: 10; IBA GmbH) inthe Fab Fragment, which is fused to the carboxy-terminus of its heavychain, as described in Example 2A. In some embodiments, a washingbuffer, as described in Example 2A, is loaded to run through the thirdselection column via operably coupled tubing. The flow-through isdirected to the waste container 10 via valves 13 operably connecting thethird selection column and waste container. In some embodiments, thewashing step is repeated a plurality of times.

The volume of affinity matrix reagent contained in the first selection,second and/or third selection columns can be the same or different, andcan be chosen based on the desired yield of the selection and/or desiredratio of CD4+ cells to CD8+ cells enriched for cells expressing a markerexpressed on central memory T (T_(CM)) cells following selection. Thevolume so-chosen is based on the assumption of an average yield of 1×10⁸cells being capable of selection per each 1 mL volume of filled column.Also, for a particular chosen culture-initiation ratio, for example, aculture-initiation ratio of 1:1 of CD4+ cells to a population containingCD8+ cells enriched for a marker expressed on central memory T (T_(CM))cells (e.g. a population of CD8+ cells enriched based on furtherselection for one of CD28, CD62L, CCR7, CD27 or CD127), the volume ofthe matrix for selecting the parent population of the enrichedpopulation, e.g. the CD8+ cells, is larger in comparison to the matrixused for selecting the other of the CD4+ or CD8+ T cell population, e.g.the CD4+ population. The amount or extent in which the volume of thematrix used to select for the parent population of the further enrichedpopulation, e.g. the matrix containing anti-CD8 Fab fragment, is greaterthan the volume of the other matrix or other matrices is chosen based onthe fraction or percentage of the further enriched population of cellsin the sample (e.g. CD8+/CD28+, CD8+/CD62L+, CD8+/CCR7+, CD8+/CD27+ orCD8+/CD127+) compared to the fraction or percentage of the parentpopulation, e.g. CD8+ cells, present in the sample. This can beestimated based on averages among patients or healthy donors, or it canbe measured for a given patient on which selection is being performedprior to determining the size of the columns to use.

In one exemplary process, the column volumes used in the process are,for example, a 2 mL column with anti-CD4 Fab fragments, a 6 mL columnwith anti-CD8 Fab fragments, and a 2 mL column with anti-CD62L Fabfragments. The column length and/or column diameter can be chosen to fitthe desired volume, for example, to achieve a desired culture-initiationratio of CD4+ cells to CD8+ cells enriched for cells expressing a markerexpressed on central memory T (T_(CM)) cells, e.g. CD8+/CD28+,CD8+/CD62L+, CD8+/CCR7+, CD8+/CD27+ or CD8+/CD127+ cells. In someembodiments, to accommodate the volume of affinity matrix, a pluralityof columns having immobilized thereto the same Fab reagent can be addeddirectly in series and operably connected to each other via a tubingline.

To achieve selection of cells, an apheresis sample is applied to thefirst selection column 1, whereby CD8+ T cells, if present in thesample, remain bound to the resin of the first column as unselectedcells (containing CD8− cells) pass through the column. The number ofcells in the starting sample used in some examples is chosen to be abovethe number of cells to be selected based on the combined volume of thematrices such as an amount greater than the capacity of the column foreach selection (e.g. an amount having greater than 200 millionCD8+/CD62L+ cells and greater than 200 million CD4+ cells, e.g. greaterby at least about 10% or 20% or greater). The flow-through containingunselected cells (negative fraction) is directed to pass to the secondcolumn 2 via a valve 13 operably connecting the first and second column.CD4+ T cells remain bound to the resin in the second column. Theflow-through containing fluid and further unselected cells (negativecells) is directed to a first waste container 10 via valves 13 operablyconnecting the second selection column and negative fraction container.

From a washing buffer reservoir 6, a washing buffer, such as describedin Example 2A, is loaded to run through the first column and the secondcolumn via a valve and tubes operably connecting the columns. Theflow-through containing any cells that are washed from the first andsecond columns is directed to the waste container 10 via valves 13operably connecting the second selection column and waste container. Insome embodiments, the washing step is repeated a plurality of times.

From an elution buffer reservoir 7, a buffer containing an eluent, suchas biotin or an analog thereof, for example 2.5 mM desthiobiotin, isloaded to run through the second column 2 via valves 13 and tubingoperably connecting the elution buffer reservoir and second column. Insome embodiments, the elution buffer contains cell culture media. Theflow-through, containing enriched CD4+ cells and residual biotin, isdirected to a removal chamber 9 to remove biotin, such as described inExample 2A, which is operably connected to the second column via a valve13 and tubing. The flow-through containing enriched cells positivelyselected for CD4+ is directed to a culture vessel 12, such as a bag, viavalves 13 operably connecting the removal chamber 9 and culture vessel.In some embodiments, the elution step is repeated. In some embodiments,after performing the elution step, an activation buffer containing aT-cell activation reagent (e.g. anti-CD3, anti-CD28, IL-2, IL-15, IL-7,and/or IL-21) replaces the wash buffer and is directed via valves andtubing from the washing buffer reservoir to the second column, removalchamber and into the culture vessel. The positive fraction collected inthe culture vessel is an isolated CD4+ cell population.

From an elution buffer reservoir 7, a buffer containing an eluent, suchas biotin, is loaded to run through the first column 1 via a valve 13and tubing operably connecting the elution buffer reservoir and firstcolumn. In some embodiments, the elution buffer contains cell culturemedia. The flow-through, containing enriched CD8+ cells and residualbiotin or an analog thereof, is directed to a removal chamber 9 toremove biotin or a biotin analog, such as described in Example 2A, whichis operably connected to the first column via a valve 13 and tubing. Theflow-through containing enriched cells positively selected for CD8+ isdirected to pass to a third column 15 via a valve 13 operably connectingthe removal chamber 9 and third column. A CD62L+ subset of the CD8+cells remain bound to the resin in the third column. The flow throughcontaining fluid and further unselected cells (negative cells) isdirected to a second waste container 11 via valves 13 operablyconnecting the third selection column and second waste container.

From a washing buffer reservoir 6, a washing buffer, such as describedin Example 2A, is loaded to run through the third column 15 via valves13 and tubing operably connecting the wash buffer reservoir and thirdcolumn. The flow through containing any cells that are washed from thethird column is directed to the second waste container 11 via valves 13operably connecting the third selection column and second wastecontainer. In some embodiments, the washing step is repeated a pluralityof times.

From an elution buffer reservoir 7, a buffer containing an eluent, suchas biotin or an analog thereof, for example 2.5 mM desthiobiotin, isloaded to run through the third column 15 via a valve 13 and tubingoperably connecting the elution buffer reservoir and third column. Insome embodiments, the elution buffer contains cell culture media. Theflow-through, containing CD8+ cells enriched for central memory T(T_(CM)) cells expressing one of CD28, CD62L, CCR7, CD27 or CD127, andresidual biotin or analog thereof, is directed to a removal chamber 9 toremove biotin or a biotin analog, such as described in Example 2A, whichis operably connected to the third column via a valve 13 and tubing. Theflow-through containing enriched T cell memory cells positively selectedfor CD8 and a marker expressed on central memory T (T_(CM)) cells, suchas one of CD28, CD62L, CCR7, CD27 or CD127, is directed to the culturevessel 12, such as a bag, via valves 13 operably connecting the removalchamber 9 and culture vessel. In some embodiments, the culture vesselcontains a T-cell activation reagent, a cell culture media, or both. Insome embodiments, the elution step is repeated. In some embodiments,after performing the elution step, an activation buffer containing aT-cell activation reagent (e.g. anti-CD3, anti-CD28, IL-2, IL-15, IL-7,and/or IL-21) replaces the wash buffer and is directed via valves andtubing from the washing buffer reservoir to the first and/or thirdcolumn, removal chamber and into the culture vessel. The positivefraction containing CD8+ cells and cells positive for a marker oncentral memory T (T_(CM)) cells, such as one of CD28, CD62L, CCR7, CD27or CD127, is collected in the culture vessel with the CD4+ positivefraction previously collected. In some embodiments, the steps of theprocess can be performed in a different order, such as by firstenriching for cells containing CD8+ cells and cells positive for amarker on central memory T (T_(CM)) cells, such as one of CD28, CD62L,CCR7, CD27 or CD127 before enriching CD4+ cells.

Example 3 Generation of a CD4+ and CD8+ T Cell Composition by SequentialPurification in a Closed System for Use in Genetic Engineering andAdoptive Cell Therapy

This example demonstrates a procedure for selecting and generating acomposition of cells containing CD4+ and CD8+ T cells, such as CD4+ andCD8+ T cells present in a culture-initiation ratio, forincubation/activation and transduction in methods associated withgenetic engineering of cells for use in connection with adoptive celltherapy.

A composition of cells generated by selection of CD4+ and CD8+ cells,performed as described in either Example 2A (CD4+ and CD8+) or Example2B (CD4+ and CD8+ enriched for CD62L+), is incubated under stimulatingconditions, such as using anti-CD3/anti-CD28 in the presence IL-2 (100IU/mL), for example, for 72 hours at 37° C. Stimulated cells then aregenetically engineered by introducing into the cells recombinant genesfor expression of recombinant antigen receptors, such as chimericantigen receptors (CARs) or recombinant TCRs, for example, by viraltransduction. In some embodiments, following the introduction, cells arefurther incubated, generally at 37 degrees C., for example, to allow forcell expansion.

The method results in an output composition with engineered CD4⁺ andCD8⁺ T cells. In some embodiments, based on the chosen volume of theselection columns, the ratio of CD4+ to CD8+ cells in the compositionthat is incubated under the stimulating conditions prior to engineering(culture-initiation ratio) results in a particular desired output ratioof CD4+ to CD8+ cells or of engineered CD4+ cells to engineered CD8+cells, or such a ratio that is within a certain range of tolerated errorof such a desired output ratio, following the incubation, stimulationand/or engineering steps. In some embodiments, such a desired outputratio or ratio within the range of tolerated error is achieved a certaintolerated percentage of the time.

Example 4 Selection of Cells Using Sub-Optimal Yield Concentrations ofFab-Coated Surfaces

Human apheresis-derived PBPC samples, at a range of different cellnumbers, were incubated in a single composition with magnetic microbeadsconjugated to anti-CD4 Fabs and magnetic microbeads conjugated toanti-CD8 Fabs, with gentle mixing for about 30 minutes. This incubationwas followed by elution of non-selected cells, and recovery usingmagnetic field carried out as described above. Incubation using between0.05 and 1.5 mL of each of microbead reagent per million cells producedlow yields of CD4+ or CD8+ cells, respectively (cells recovered for therespective selection compared with positive cells in the incubation),which in this study were generally in the range of 15-70%). On average,greater yields were observed at higher concentrations of reagents percell using such suboptimal yield concentrations.

Accordingly, in one example, a selection is carried out, in which PBMCsare incubated with suboptimal concentrations per cell number of beadscoupled to a CD4-binding and beads coupled to a CD8-binding agent, andcells recovered using a magnetic field. Based on the observedrelationship between concentration of the reagent per cell and yield forthe beads used, one or the other of the CD4-binding or CD8-bindingreagent is included at a higher concentration (e.g., higher number ofCd4 or CD8 binding molecules included per cell), thereby producing anoutput ratio of CD8+:CD4+ cells following selection, which is greaterthan or less than 1, such as 1.5:1 or 2:1 or 3:1 or 1:1.5, 1:2, or 1:3.The output ratio may be selected based on the desired ratio of CD8 vs.CD4 cells in a composition containing engineered cells to be producedfollowing one or more additional steps, such as activation,transduction, and/or expansion.

What is claimed:
 1. A method for producing genetically engineered Tcells, the method comprising: (a) performing a first selection in aclosed system, said first selection comprising enriching for one of (i)CD4⁺ cells and (ii) CD8+ cells from a sample containing primary human Tcells, the enrichment thereby generating a first selected population anda non-selected population, wherein the sample is a blood orblood-derived sample from a subject with cancer and comprises at least1×10⁸ cells; (b) performing a second selection in the closed system,said second selection comprising enriching for the other of (i) CD4+cells and (ii) CD8+ cells from the non-selected population, theenrichment thereby generating a second selected population, (c)combining the cells of the first selected population and cells of thesecond selected population at a ratio of CD4+ cells to CD8+ cells ofbetween 2:1 and 1:2 to produce a culture-initiating composition; (d)incubating the culture-initiating composition, comprising cells of thefirst selected population and cells of the second selected population ina culture vessel under stimulating conditions, thereby generatingstimulated cells; (e) introducing a genetically engineered antigenreceptor into a plurality of the stimulated cells generated in (d),wherein the antigen receptor binds to an antigen expressed by cells ofthe cancer; and (f) incubating the genetically engineered cellsgenerated in (e) to allow for expansion, wherein the method generates anoutput composition comprising CD4+ T cells and CD8+ T cells expressingthe genetically engineered antigen receptor, and the output compositionhas a ratio of CD4+ cells to CD8+ cells that is between 5:1 and 1:5. 2.The method of claim 1, wherein said combining is performed in the closedsystem.
 3. The method of claim 1, wherein the sample is obtained from ahuman subject, and the ratio of CD4+ cells to CD8+ cells in theculture-initiating composition is at least 10%, greater or at least 10%less than the ratio of CD4+ cells to CD8+ cells in the sample.
 4. Themethod of claim 1, wherein the enriching cells in the first selectionand the second selection comprises immunoaffinity-based selection. 5.The method of claim 4, wherein the first immunoaffinity-based selectionand the second immunoaffinity-based selection comprise: contacting cellswith an antibody capable of specifically binding to a cell surfacemarker; and recovering cells bound to the antibody, thereby effectingpositive selection, or recovering cells not bound to the antibody,thereby effecting negative selection, wherein the recovered cells areenriched for the CD4+ cells or the CD8+ cells.
 6. The method of claim 1,wherein the first selection and the second selection are carried out inseparate separation vessels, which are operably connected.
 7. The methodof claim 4, wherein the immunoaffinity-based selection in the firstselection and the immunoaffinity-based selection in the second selectionindependently each comprises: contacting cells with an antibodyimmobilized on or attached to an affinity chromatography matrix, saidantibody capable of specifically binding to a cell surface marker toeffect positive or negative selection of CD4+ cells or CD8+ cells; andrecovering cells from the affinity chromatography matrix enriched forthe CD4+ cells or the CD8+ cells.
 8. The method of claim 7, wherein: theantibody comprises one or more binding partners capable of forming areversible bond with a binding reagent immobilized on the affinitychromatography matrix, wherein the antibody is reversibly bound to saidmatrix during said contacting; and cells expressing a cell surfacemarker specifically bound by the antibody on said affinitychromatography matrix are capable of being recovered from the affinitychromatography matrix by disrupting the reversible binding between thebinding reagent and binding partner.
 9. The method of claim 8, wherein:the binding partner is selected from the group consisting of biotin, abiotin analog, and a peptide capable of binding to the binding reagent;and the binding reagent is selected from among the group consisting ofstreptavidin, a streptavidin mutein that binds biotin or a streptavidinbinding peptide, avidin, and an avidin mutein that binds biotin or astreptavidin binding peptide.
 10. The method of claim 8, whereindisrupting the reversible binding comprises applying a competitionreagent to disrupt the bond between the one or more binding partners andthe binding reagent, thereby recovering the selected cells from thematrix.
 11. The method of claim 7, wherein the affinity chromatographymatrix of the first selection and the affinity chromatography matrix ofthe second selection are packed in a separation vessel, which is acolumn.
 12. The method of claim 7, wherein the affinity chromatographymatrix adsorbs and/or is capable of selecting at least or at least about50×10⁶ cells/mL.
 13. The method of claim 1, wherein the enriching forthe CD4+ cells comprises positive selection based on surface expressionof CD4 and the enriching for the CD8+ cells comprises positive selectionbased on surface expression of CD8.
 14. The method of claim 1, whereinthe one of the first selection and the second selection that comprisesenriching for the CD8+ cells further comprises enriching for centralmemory T (T_(CM)) cells.
 15. The method of claim 1, wherein the ratio ofCD4+ cells to CD8+ cells in the culture-initiating composition producedby step (c) is different than the ratio of CD4+ cells to CD8+ cells inthe sample.
 16. The method of claim 5, wherein the antibody isimmobilized on the outside surface of a bead or a particle.
 17. Themethod of claim 16, wherein: the antibody comprises one or more bindingpartners capable of forming a reversible bond with a binding reagentimmobilized on the surface, whereby the antibody is reversiblyimmobilized to said surface; and the method comprises after contactingthe cells in the sample to the antibody of the first immunoaffinitybased selection and the second immunoaffinity based selection, applyinga competition reagent to disrupt the bond between the binding partnerand binding reagent, thereby recovering the selected cells bound to theantibody.
 18. The method of claim 17, wherein: the binding partner isselected from among the group consisting of biotin, a biotin analog, anda peptide capable of binding to the binding reagent; and the bindingreagent is selected from among the group consisting of streptavidin, astreptavidin mutein that binds biotin or a streptavidin binding peptide,avidin, and an avidin mutein that binds biotin or a streptavidin bindingpeptide.
 19. The method of claim 1, wherein the output compositioncomprises a ratio of CD4+ cells to CD8+ cells that is between 3:1 and1:3.
 20. The method of claim 1, wherein the stimulating conditions in(d) comprise the presence of an anti-CD3 antibody and an anti-CD28antibody.
 21. The method of claim 1, wherein the stimulating conditionsin (d) comprise an agent capable of activating one or more intracellularsignaling domains of one or more components of a TCR complex.
 22. Themethod of claim 1, wherein: the genetically engineered antigen receptorcomprises a T cell receptor (TCR) or a functional non-TCR antigenreceptor; or the genetically engineered antigen receptor comprises achimeric antigen receptor (CAR).
 23. The method of claim 1, wherein theratio of CD4+ cells to CD8+ cells in the culture-initiating compositionis 1:1.
 24. The method of claim 21, wherein the stimulating conditionsin (d) comprise the presence of one or more of an anti-CD3 antibody, ananti-CD28 antibody, an anti-4-1BB antibody, and a cytokine.
 25. Themethod of claim 1, wherein the one of the first selection and the secondselection that comprises enriching for the CD8+ cells further comprisesenriching for cells expressing a marker selected from the groupconsisting of CD28, CD62L, CCR7, CD127 and CD27.
 26. The method of claim1, wherein the sample is an apheresis sample, a peripheral bloodmononuclear cell (PBMC) sample, or a leukapheresis sample.
 27. Themethod of claim 1, wherein the first selected population and/or thesecond selected population comprises at least 1×10⁶ cells.
 28. Themethod of claim 1, wherein the first selected population and/or thesecond selected population comprises at least 1×10⁷ cells.